AIPA, OMPA, and ASP14 in vaccine compositions and diagnostic targets for anaplasma phagocytophilum infection

ABSTRACT

Anaplasma phagocytophilum surface protein AipA and/or fragments thereof which comprise an invasin domain are used in compositions suitable to elicit an immune response to treat or prevent infections caused by tick-born bacteria of the Anaplasmatacaea family. AipA proteins and protein fragments or antibodies directed to AipA proteins and protein fragments are also used in diagnostic assays to detect exposure to and/or infection with Anaplasmatacaea. AipA and/or fragments thereof are also used for these purposes in combination with one or both of Asp14 and OmpA proteins and/or fragments thereof which comprise an invasin domain. Homologs of these proteins are also used in the compositions and assays.

FIELD OF THE INVENTION

The invention generally relates to a vaccine or immunogen and diagnostic for anaplasmosis in mammals. In particular, the invention provides Anaplasma phagocytophilum (AipA) protein and fragments thereof comprising an invasin domain for these purposes, used alone or in combination with one or both of Anaplasma phagocytophilum outer surface protein A (OmpA) and/or antigenic fragments thereof, and Anaplasma phagocytophilum surface protein 14 (Asp14) and/or antigenic fragments thereof.

PRIORITY

This application claims the benefit of U.S. application 61/935,012, filed Feb. 3, 2014. This application is incorporated herein by reference in its entirety.

SEQUENCE LISTING

This document incorporates by reference an electronic sequence listing text file, which was electronically submitted along with this document. The text file is named 02940981TA_ST25.txt, is 108 kilobytes, and was created on Jan. 30, 2015.

BACKGROUND OF THE INVENTION

Anaplasma phagocytophilum (Aph) is a tick-transmitted obligate intracellular bacterium of the family Anaplasmataceae that can infect humans, livestock, companion animals, and wild animals. In addition to Aph, the Anaplasmataceae family members include Anaplasma marginale, Anaplasma platys, Ehrlichia chaffeensis, Ehrlichia canis, and Ehrlichia ruminatium, among others, and all of these cause similar infections known collectively as ehrlichiosis or anaplasmosis. When humans contract an Aph infection, it is more specifically known as human granulocytic anaplasmosis (HGA) and is marked by fever and increased susceptibility to potentially fatal opportunistic infections. Other possible disease manifestations include leukopenia, thrombocytopenia, and elevated serum transaminase levels.

Unfortunately, no vaccines against these infectious agents are currently available. Instead, antibiotic treatments with doxycycline or tetracycline are currently the frontline treatments for anaplasmosis/ehrlichiosis and can be effective. However, the overuse of antibiotics is currently of great concern, and many individuals, such as those that are allergic to the drug, pregnant women, and small children, either cannot or are not advised to take doxcycline. Also troubling is the lack of tools for rapid and definitive diagnosis of anaplasmosis/ehrlichiosis in any species other than dogs.

HGA is an emerging and potentially fatal disease transmitted by the same vectors that transmit Lyme Disease, primarily ticks and deer, but other animal and human hosts can complete the vector cycle and therefore extend the spread of disease. In the U.S., HGA occurs primarily in the Northeast, Upper Midwest, and Northern California. Since HGA became a reportable disease in the U.S. in 1999, the number of cases has risen annually, reaching 2,037 in 2013. Since diagnostic tools for HGA are lacking, the number of actual cases is likely to be much higher. HGA is increasingly recognized in Europe and Asia, and Aph infection is now the most widespread tick-transmitted disease of animals in Europe. Domestic animals, such as dogs and cats, and livestock, such as sheep can be become infected.

As the name implies, an obligate intracellular bacterium must enter a target cell in its human or animal host to survive, replicate, and move to the next host. When an Anaplasma spp. or Ehrlichia spp. infected tick bites a subject, the bacteria is transferred from the tick salivary glands into the tissues of the host or into the bloodstream where they bind to the surface of host cells (especially neutrophils) and are internalized by being taken up into vacuoles that form around each bacterium. For A. phagocytophilum, optimal invasion of mammalian host cells is known to involve multiple cell surface proteins (various adhesins and invasins) which function cooperatively. Within the cell, a resident bacterium prevents the vacuole from merging with lysosomes, which would otherwise destroy the bacterium. In doing so, the bacterium converts the cell into a protective niche that favors bacterial survival and completion of its zoonotic cycle. For example, A. phagocytophilum undergoes a biphasic developmental cycle within neutrophils in which it transitions between an infectious morphotype (dense-cored cell; DC) and a non-infectious, replicative morphotype (reticulate cell; RC). Subsequently, if the Aph infected host is bitten by a tick, DC cells from the host can be ingested by the tick during the blood meal, and the disease is then transferred to new hosts when the infected tick takes another blood meal. However, HGA can also be transmitted perinatally and by blood transfusion, and possibly nosocomially.

While the hallmark of Aph infection is Aph colonization of neutrophils, Aph has also been detected in the microvascular endothelium of heart and liver in experimentally infected severe combined immunodeficiency mice. For experimental purposes, promyelocytic and endothelial cell lines are also useful as in vitro models for studying Aph-host cell interactions. It has been shown that when naïve neutrophils or HL-60 cells are overlaid on Aph-infected endothelial cells, the bacterium rapidly transmigrates into the myeloid cells, demonstrating that Aph infects endothelial cells in vitro and suggesting that the bacterium may transmigrate between endothelial cells and neutrophils during the course of mammalian in vivo infections.

As noted above, humans are not the only species that are infected by members of the Anaplasmataceae family. For example, A. marginale infects bovine erythrocytes. Bovine anaplasmosis causes severe economic loss in many countries, mainly due to the high morbidity and mortality in susceptible cattle herds. Several parameters contribute to the losses due to anaplasmosis and include: low weight gain, reduction in milk production, abortion, the cost of anaplasmosis treatments, and mortality. The losses incurred by the U.S. cattle industry as a result of anaplasmosis are estimated to be $300 million per year whereas in Latin America, losses are calculated to be approximately $800 million per year.

Proteins which mediate the infection of endothelial cells and/or the dissemination of bacteria into the microvasculature of heart and liver are attractive targets for preventing disease caused by Anaplasmataceae bacteria such as Aph. In particular, targeting Aph proteins that are conserved among related Anaplasmataceae family members may also reduce or block transmission of disease caused by a broad spectrum of Anaplasmataceae species. In addition, it would be advantageous to have available a rapid and highly accurate diagnostic test for detecting Anaplasmataceae species infection, especially one that allowed detection early in infection. Unfortunately, no FDA-approved vaccines against Anaplasmataceae species having purified antigens are currently available.

The current gold standard serologic test for diagnosis of anaplasmosis in humans is indirect immunofluorescence assays (IFA) performed as timed pairs over a period of a few weeks, only available in specialized reference laboratories. This assay measures non-specific increases in IgM and IgG antibody levels. However, IgM antibodies, which usually rise at the same time as IgG near the end of the first week of illness and remain elevated for months or longer, are even less specific than IgG antibodies and more likely to result in a false positive. Serologic tests based on enzyme immunoassay (EIA) technology are available from some commercial laboratories. However, EIA tests are qualitative rather than quantitative, meaning they only provide a positive/negative result, and are less useful to measure changes in antibody titers between paired specimens. Furthermore, some EIA assays rely on the evaluation of IgM antibody alone, which again may have a higher frequency of false positive results. Between 5-10% of currently healthy people in some areas may have elevated antibody titers due to past exposure to Aph or to other Anaplasmataceae family members. If only one sample is tested, it can be difficult to interpret. A four-fold rise in antibody titer is needed to achieve significance in paired samples taken weeks apart. Thus, tools for a rapid and definitive diagnosis in any species other than dogs are lacking.

U.S. Pat. No. 7,906,296 B2 to Beall et al teaches polynucleotide sequences from major outer surface protein P44 of Anaplasma platys (Apl), which causes tick-born anaplasmosis in dogs. P44 and peptides from the translated protein can be used for detection of Apl and Aph infection and/or to elicit an immune response in vivo and confer resistance to anaplasmosis caused by Apl or Aph.

U.S. Pat. Nos. 8,158,370; 8,303,959 and US 2013/0064842, all to Liu et al, teach the use of P44 surface proteins from various strain variants of Aph to diagnose and protect against anaplasmosis.

U.S. Pat. No. 8,609,350 B2 to Liu et al. teaches polypeptides from Aph to diagnose and protect against anaplasmosis. The Aph sequences were derived from APH_0915, which encodes a hypothetical open reading frame of a protein of unknown function.

PCT/US2013/047325 to Carlyon, which is herein incorporated by reference, relates to the invasin proteins OmpA and Asp14, and fragments thereof comprising conserved invasin domains. The proteins and fragments are used as therapeutic and diagnostic agents for A. phagocytophilum infection.

Nelson, C. M., et al. (BMC Genomics 9, 364; 2008) used a whole genome transcriptional profiling tiling array analysis to detect A. phagocytophilum genes that are upregulated in vitro during infection of mammalian versus tick cell lines. Many of the proteins encoded by the identified genes were assigned a designation of “hypothetical protein” and of these, several had a predicted cellular location of “outer membrane”, among them APH0915. However, no further information about this putative protein was provided, and no confirmation of its status as an outer membrane protein or possible significance was shown.

A need remains in the art for immunogenic compositions and vaccines to combat Anaplasmataceae infections and for methods to rapidly and accurately diagnose new cases of these diseases, especially with respect to Anaplasmataceae infections that cause anaplasmosis and HGA.

SUMMARY OF THE INVENTION

Obligate intracellular bacteria such as Anaplasmataceae use outer membrane proteins called invasins to enter and infect eukaryotic host cells. Since these organisms are incapable of extracellular survival, blocking this internalization step prevents infection and transmission of diseases caused by Anaplasmataceae. Thus, the identification and characterization of Anaplasmataceae invasin proteins provides a path forward for the development of therapies to treat and/or prevent Anaplasmataceae infections. In addition, the discovery and characterization of such invasins also leads to improved diagnostic methods for detecting exposure to and/or infection with Anaplasmataceae, so that proper therapeutic measures can be undertaken.

Provided herein is a newly characterized invasin protein from A. phagocytophilum, denoted AipA (Anaplasma phagocytophilum invasion protein A) (SEQ ID NO: 13). This protein corresponds to protein APH0915 identified by Nelson et al. (2008). The studies presented in Example 1 below demonstrate that AipA is in fact an outer membrane protein, and further that AipA is, surprisingly, an A. phagocytophilum invasin protein that is useful as a therapeutic and diagnostic agent. In addition, the detailed characterization of AipA presented herein has identified particular fragments of AipA which constitute domains required for the protein to function as an invasion, so-called effector or invasin domains, that are also useful as therapeutic and diagnostic agents. Further, AipA is unique to A. phagocytophilum i.e. homologs of AipA are not present in other Anaplasmataceae species. Accordingly, certain aspects of this disclosure provide AipA and/or one or more functional fragments thereof for use as components of vaccines and/or immunogenic compositions to prevent or treat A. phagocytophilum infections. Other aspects provide AipA protein and fragments thereof comprising invasin domains, and antibodies thereto, as diagnostic targets to detect exposure to and/or infection with A. phagocytophilum. Exemplary functional domains of AipA include but are not limited to fragments which include a linear amino acid sequence encompassing amino acids 1-87 inclusive, and amino acids 9-21 (SEQ ID NO: 13), inclusive.

In other aspects, AipA and/or one or more of the identified functional fragments thereof are used for these purposes in combination with one or more additional invasins and/or functional domains of the additional invasins. Exemplary additional invasins include but are not limited to Asp14 (14-kDa A. phagocytophilum surface protein; APH0248) and OmpA (Outer membrane protein A; APH0338 in the annotated A. phagocytophilum proteome), and homologs thereof from other species, e.g. other Anaplasmataceae species. Exemplary functional domains of Asp 14 include but are not limited to fragments which include the linear amino acid sequence from amino acids: 19-60, 101-112, 101-124, and 113-124, all of which are inclusive. In some aspects, the sequence is that which encompasses amino acids 113-124. Exemplary functional domains of OmpA include but are not limited to fragments which include the amino acid sequence from amino acids: 19-74, 59-74, 48-56, 86-101, 135-155, and 24-121, all of which are inclusive. In some aspects, the sequence is that which encompasses amino acids 59-74. Accordingly, in some aspects, what is provided herein are various combinations of these three A. phagocytophilum surface proteins and/or invasion domain-containing fragments thereof, as well as antibodies to one or more of these proteins or fragments, as protective/therapeutic/diagnostic agents or targets to treat or detect Anaplasmataceae infection.

Significantly, it has been discovered that these three invasins work together synergistically to promote infection of host cells, and that combinations of two of AipA, Asp14 and OmpA proteins (or peptides comprising invasin domains thereof) promote infection more effectively than any one alone, and, in some aspects, the most effective combination utilizes all three. Conversely, to prevent infection of host cells from infection, some aspects of the invention provide therapeutic agents which are or which are based on combinations of at least two and usually all three of the proteins or relevant fragments thereof. For example, linear peptide domains of each protein that mediate invasin and combinations of linear peptide domains, are encompassed, as are antibodies targeting one, two, or all three domains. Such agents are shown herein to simultaneously work synergistically to nearly abolish A. phagocytophilum invasion of host cells. For example, antisera combinations targeting two of the three invasins together more effectively inhibited infection than an antiserum targeting an individual protein, and the most effective blocking of A. phagocytophilum infection was achieved using antisera against all three invasins.

An embodiment of the invention is a composition comprising one or more polypeptides which include one copy or more than one copy of SEQ ID NO:14 together with one copy or more than one copy of either or both of SEQ ID NO: 3 and SEQ ID NO: 06. For example, a single polypeptide might include 1, 2, or 3 copies of SEQ ID NO: 14 together with 0, 1, 2 or 3 copies of SEQ ID NO. 3 and/or 0, 1, 2 or 3 copies of SEQ ID NO. 6, and may include additional amino acid sequences of interest (e.g. linkers, etc.). Or the composition may include two or more polypeptides wherein one polypeptide is or includes one or more copies of SEQ ID No. 14, and the other polypeptide(s) is/are or include(s) one or more copies of either or both of SEQ ID No. 3 and SEQ ID No. 6.

In exemplary embodiments, the isolated peptides are in a vehicle or carrier suitable for administration to a subject. At least one of the polypeptides may be linked to an amino acid spacer, an amino acid linker, a signal sequence, a stop transfer sequence, a transmembrane domain, a protein purification ligand, a heterologous protein, or one or more additional polypeptides comprising SEQ ID NO:01, 02, 03, 04, 05, 06, 13, 14, or 15 or a combination thereof. An exemplary embodiment is one or more polypeptides linked to a protein purification ligand, and protein purification ligands are a peptide encoding a histidine tag.

Another embodiment of the invention comprises one or more polypeptides selected from the group consisting of SEQ ID NO:01, SEQ ID NO:02, and SEQ ID NO:03. Another embodiment of the invention comprises one or more polypeptides selected from the group consisting of SEQ ID NO:04, SEQ ID NO:05, and SEQ ID NO:06. Another embodiment of the invention comprises one or more polypeptides selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

Another embodiment of the invention includes nucleic acids and nucleic acid based compositions that can be used for generating an immune response. In particular embodiments, the compositions may take the form of DNA vaccines or other nucleic acid vaccines which comprise nucleic acids encoding one or more polypeptides as described herein, e.g. polypeptides which include one or more copies of SEQ ID NO:14, optionally together with one or more copies of either or both SEQ ID NO: 3 and SEQ ID NO. 06. For example, a suitable composition might include a single nucleic acid in a vector (e.g., adenovirus, baculovirus, etc.) that codes for a single polypeptide or multiple polypeptides that include 1, 2, or 3 copies of SEQ ID NO: 14, together with 0, 1, 2 or 3 copies of SEQ ID NO. 3; and 0, 1, 2 or 3 copies of SEQ ID NO. 6, and may include additional amino acid sequences of interest, linkers, etc. Or the composition may include two or more nucleic acids, each on a vector, which code for a polypeptide that is or includes one or more repeats of SEQ ID NO. 14, and code for another polypeptide is or includes one or more repeats of either or both SEQ ID NO. 3 and SEQ ID NO. 6. DNA may also be provided without a vector and be combined with material which might also be driven into a cell or living tissue by an agent which crosses into a cell (e.g., Lipofectamine). Furthermore, the DNA vaccines might be incorporated into a host bacterium that is itself administered as a vaccine to a subject.

Another embodiment of the invention is a method of protecting a subject from acquiring a zoonotic disease and/or treating a zoonotic disease in a subject by the step of administering a composition an immunogenic composition comprising the isolated polypeptide represented by SEQ ID NO:14 and at least one of the isolated polypeptides represented by SEQ ID NO:03 and SEQ ID NO:06. At least one of the polypeptides may be linked to an amino acid spacer, an amino acid linker, a signal sequence, a stop transfer sequence, a transmembrane domain, a protein purification ligand, a heterologous protein, or one or more additional polypeptides comprising SEQ ID NO:01, 02, 03, 04, 05, 06, 13, 14, or 15; or a combination thereof. The zoonotic disease may be one caused by an obligate intracellular Anaplasmataceae bacterium selected from the group consisting of Anaplasma phagocytophilum, Anaplasma marginale, Anaplasma platys, Ehrlichia chaffeensis, Ehrlichia canis, Ehrlicia ruminantium, and Ehrlichia ewingii. When the subject is a human, the zoonotic disease may be human granulocytic anaplasmosis (HGA) if caused by Anaplasma phagocytophilum. When the subject is an animal, the zoonotic disease may be anaplasmosis. If caused by an ehrlichia species, the human zoonotice disease may be monocytic ehrlichiosis or ehrlichiosis.

Another embodiment of the invention is a method of detecting antibodies that specifically bind an Anaplasmataceae polypeptide in a test sample. The method may include the steps of contacting a test sample, under conditions that allow polypeptide-antibody complexes to form, with a composition that includes at least one or more polypeptides encoding all or a portion of SEQ ID NO:01, SEQ ID NO:04, and/or SEQ ID NO:13, and detecting said polypeptide-antibody complexes, wherein the detection is an indication that antibodies specific for Anaplasmataceae Asp14, OmpA, or AipA are present in the test sample. Alternatively, antibodies are generated to at least one or more polypeptides encoding all or a portion of SEQ ID NO:01, SEQ ID NO:04, and/or SEQ ID NO:13, and the antibodies are used to detect the presence of AipA and/or OmpA and/or Asp14 protein in a biological sample from a subject. The method may be an assay selected from the group consisting of an immunoblot and an enzyme-linked immunosorbant assay (ELISA). An exemplary embodiment of this methodology includes using at least one polypeptide which is or includes SEQ ID NO:03, SEQ ID NO:06, or SEQ ID NO:14 in the assay, whereby infection with obligate intracellular Anaplasmataceae is determined from a serum sample exhibiting antibody binding with the at least one polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B. Schematic diagrams of A. phagocytophilum AipA membrane topology and sequence. (A) Diagram of the predicted topology of AipA in the A. phagocytophilum outer membrane. N, AipA amino terminus. C, AipA carboxy terminus. Numerical values indicate amino acid coordinates for predicted transmembrane spanning regions. (B) Diagrams of the AipA sequence. The scale indicates 50-amino acid intervals. For the Hydrophobicity diagram, the Kyte-Doolitle algorithm was used to determine hydrophobic (histogram above the axis) and hydrophilic (histogram below axis) regions. For the Surface diagram, the Emini algorithm was used to determine regions that are likely accessible on the surface of the AipA (histogram above the axis) or not (histogram below the axis). The AipA amino acid segments against which antisera were raised are indicated on the Hydrophobicity plot by horizontal lines.

FIG. 2A-F. Differential expression profiling of AipA throughout the A. phagocytophilum life cycle. (A) aipA transcriptional profile during A. phagocytophilum infection of HL-60 cells. DC bacteria were incubated with HL-60 cells to establish a synchronous infection. Total RNA isolated from the DC inoculum and infected host cells at several postinfection time points was subjected to reverse transcriptase-quantitative PCR (RT-qPCR). Relative aipA transcript levels were normalized to A. phagocytophilum 16s rRNA gene transcript levels. To determine relative aipA transcription between RC and DC organisms, normalized aipA transcript levels per time point were calculated as the fold change in expression relative to expression at 16 h, a time point at which the entire bacterial population is in the DC morphotype. Data are the means±standard deviations (SD) for triplicate samples and are representative of two experiments having similar results. (B) Western blot screening of whole-cell lysates of uninfected (U) and A. phagocytophilum infected HL-60 cells (I) using mouse antiserum raised against GST-AipA₁₋₈₇ (αAipA₁₋₈₇) and GST-AipA₂₄₉₋₃₅₅ (αAipA₂₄₉₋₃₅₅). (C) AipA expression over the course of infection of mammalian host cells. RF/6A cells that had been synchronously infected with A. phagocytophilum (Ap) were screened with antibodies targeting Msp2 (P44) (to denote all A. phagocytophilum inclusions) and AipA viewed by confocal microscopy. Data presented are the mean percentages±SD of Msp2 (P44)-positive A. phagocytophilum inclusions that were also AipA-positive. At least 100 bacterial inclusions were scored per time point. (D) aipA and groEL expression during transmission feeding of A. phagocytophilum infected ticks on naïve mice. A. phagocytophilum-infected I. scapularis nymphs were allowed to feed on mice for 72 h. Total RNA recovered from unfed and transmission-fed ticks that had been removed at 24, 48, and 72 h postattachment was subjected to RT-qPCR. Relative aipA and groEL transcript levels were normalized to A. phagocytophilum 16S rRNA gene levels. (E) AipA is not expressed during A. phagocytophilum infection of a tick cell line. Western blot analysis of uninfected and A. phagocytophilum infected (Inf.) HL-60 and ISE6 cells using antiserum specific for AipA₁₋₈₇ or APH0032. The number of weeks (Wk.) during which A. phagocytophilum was maintained in ISE6 cells are indicated. (F) AipA is expressed in vivo and elicits a humoral immune response. Western blot analysis of GST and GST-AipA₁₋₈₇ screened with sera from an HGA patient and from an A. phagocytophilum infected dog. Results presented in panels B to F are each representative of at two to three independent experiments with similar results. Statistically significant (*, P<0.05; **, P<0.005; ***, P<0.001) values are indicated.

FIG. 3A-B. AipA is an A. phagocytophilum OMP. (A) AipA colocalizes with the confirmed OMP, Msp2 (P44). A. phagocytophilum-infected RF/6A cells were fixed and viewed by confocal microscopy to assess immunoreactivity with AipA antiserum in conjunction with Msp2 (P44) antiserum. Host cell nuclei were stained with DAPI (4′,6′-diamidino-2-phenylindole). The insets demarcated by solid boxes in the lower right corners of each panel are magnified versions of the representative A. phagocytophilum-occupied vacuole that is denoted by the hatched box in each panel. (B) AipA is exposed on the bacterial surface. Intact A. phagocytophilum DC organisms were incubated with trypsin or vehicle control, solubilized, and Western-blotted. Immunoblots were screened with antiserum targeting AipA₁₋₈₇, Asp55, or APH0032. Data are representative of two experiments with similar results.

FIG. 4A-F. GST-AipA requires amino acids 1 to 87 to bind and competitively inhibit A. phagocytophilum infection of mammalian host cells. (A and B) GST-AipA₁₋₈₇ binds to mammalian host cells. RF/6A cells were incubated with GST-AipA₁₋₈₇, GST AipA₂₄₉₋₃₅₅, or GST alone. (A) The host cells were fixed, screened with GST antibody, and examined using confocal microscopy. Host cell nuclei were stained with DAPI. Representative merged fluorescent images from three experiments with similar results are shown. (B) Flow cytometric analysis of GST fusion protein binding to RF/6A cells. (C to F) GST-AipA₁₋₈₇ competitively inhibits A. phagocytophilum infection. HL-60 (C and D) and RF/6A cells (E and F) were incubated with DC bacteria in the presence of GST, GST-AipA₁₋₈₇, or GST-AipA₂₄₉₋₃₅₅ for 1 h. Following removal of unbound bacteria, host cells were incubated for 24 h (C and D) or 48 h (E and F) and subsequently examined using confocal microscopy to assess the percentage of infected cells (C and E) or the mean number (±SD) of pathogen-occupied vacuoles per cell (D and F). Results shown are relative to GST-treated host cells and are the means±SD for three experiments. Statistically significant (*, P<0.05; **, P<0.005; ***, P<0.001) values are indicated.

FIG. 5A-C. Pretreatment of A. phagocytophilum with AipA₁₋₈₇ antiserum inhibits infection of HL-60 cells but does not alter binding to sLe^(x)-capped PSGL-1. A. phagocytophilum DC organisms were exposed to antiserum targeting AipA₁₋₈₇, AipA₂₄₉₋₃₅₅, OmpA, or preimmune serum and then incubated with HL-60 (A and B), PSGL-1 CHO cells, or untransfected CHO cells (C). The infection of HL-60 cells was allowed to proceed for 24 h prior to being assessed, while bacterial binding to PSGL-1 CHO cells was assessed immediately. The mean±standard deviations of percentages of infected HL-60 cells (A), A. phagocytophilum (Ap) vacuolar inclusions per HL-60 cell (B), and bound DC organisms per PSGL-1 CHO cell or untransfected CHO cell (C) were determined using immunofluorescence microscopy. Additional positive controls for blocking A. phagocytophilum to PSGL-1 CHO cells, besides incubating bacteria with OmpA antiserum, were PSGL-1 CHO cells that had been incubated with PSGL-1 N-terminus blocking antibody KPL-1 or sLe^(x)-blocking antibody CSLEX1 prior to the addition of bacteria. Negative controls were PGSL-1 CHO cells that had been incubated with isotype control antibodies prior to the addition of bacteria. Results shown are relative to GST-treated host cells and are the means±SD for three experiments. Statistically significant (***, P<0.001) values are indicated.

FIG. 6A-B. A combination of antisera targeting AipA, OmpA, and Asp14 blocks A. phagocytophilum infection of host cells. DC bacteria were incubated with preimmune serum or antiserum targeting AipA₁₋₈₇, OmpA, and/or Asp14 and then incubated with HL-60 cells. (A) The cells were fixed and screened by confocal microscopy to assess the percentage of infected cells. Results shown are relative to host cells that had been treated with preimmune serum and are the means±SD for three experiments. (B) DNA isolated from the cells was subjected to quantitative PCR analyses. Relative DNA loads of A. phagocytophilum 16s rRNA gene were normalized to DNA loads of the human β-actin gene. Results shown are the means±SD of triplicate samples and are representative of three independent experiments with similar results. Statistically significant (*, P<0.05; **, P<0.005; ***, P<0.001) values relative to the bacterial load of host cells that had been incubated with preimmune antisera are presented.

FIG. 7A-C. AipA residues 9-21 are critical for establishing infection in host cells. (A) Western blot analyses in which rabbit antiserum targeting AipA₉₋₂₁, AipA₆₁₋₈₄, AipA₁₆₅₋₁₈₂, AipA₁₈₃₋₂₀₁, AipA₁₋₈₇, or preimmune rabbit serum was used to screen whole cell lysates of uninfected (U) and A. phagocytophilum infected HL-60 cells (I). Data are representative of two experiments with similar results. (B) ELISA in which AipA₉₋₂₁, AipA₆₁₋₈₄, AipA₁₆₅₋₁₈₂ and AipA₁₈₃₋₂₀₁ antibodies were used to screen wells coated with peptides corresponding to AipA residues 9-21, 61-84, 165-182 and 183-201. Each antiserum only recognized the peptide against which it had been raised. Results shown are the mean (±SD) of triplicate samples. Data are representative of three experiments with similar results. (C) Pretreatment of A. phagocytophilum with AipA₉₋₂₁ antiserum inhibits infection of HL-60 cells. DC bacteria were pretreated with antiserum specific for AipA₉₋₂₁, AipA₆₁₋₈₄, AipA₁₆₅₋₁₈₂, AipA₁₈₃₋₂₀₁, AipA₂₄₉₋₃₅₅, OmpA, or preimmune serum for 30 min. Next, the treated bacteria were incubated with HL-60 cells for 60 min. After removal of unbound bacteria, host cells were incubated for 24 h and subsequently examined using Msp2 (P44) antibody and confocal microscopy to assess the percentage of infected cells. Results shown are relative to preimmune serum-treated host cells and are the means±SD for six experiments. Statistically significant (*, P<0.05; **, P<0.005; ***, P<0.001) values are indicated.

FIG. 8A-C. OmpA amino acids 59 to 74 are critical for A. phagocytophilum to bind to sLe^(x)-capped PSGL-1 and for infection of mammalian host cells. (A) ELISA in which OmpA23-40, OmpA41-58, and OmpA59-74 antibodies (diluted 1:1600) were used to screen wells coated with GST, GST-OmpA, GST-OmpA19-74, GST-OmpA75-205, or peptides corresponding to OmpA23-40, OmpA41-58, or OmpA59-74. Results shown are the mean±SD of triplicate samples and are representative of three independent experiments with similar results. (B) Pretreatment of A. phagocytophilum with OmpA59-74 antibody inhibits infection of HL-60 cells in a dose-dependent manner. DC bacteria were incubated with 200 μg/ml of preimmune serum, 200 μg/ml of serum raised against GST-OmpA, or twofold serially-diluted concentrations of sera raised against OmpA23-40, OmpA41-58, or OmpA59-74 ranging from 0 to 200 μg/ml and then incubated with HL-60 cells. The infection was allowed to proceed for 24 h after which the cells were fixed and examined using immunofluorescence microscopy to quantify the percentage of infected cells. Results shown are relative to host cells that had been incubated with bacteria exposed to preimmune serum and are representative of three experiments with similar results. (C) OmpA59-74 antibody inhibits A. phagocytophilum binding to sLe^(x)-capped PSGL-1. DC bacteria were exposed to preimmune serum, antibodies against OmpA, OmpA23-40, OmpA41-58, or OmpA59-74 and then incubated with PSGL-1 CHO cells. Bacteria that were not exposed to antibodies and incubated with PSGL-1 CHO cells or CHO (−) cells were positive and negative controls, respectively, for bacterial binding. The mean numbers±SD of bound DC organisms per cell were determined using immunofluorescence microscopy. Results shown are the mean±SD of six combined experiments. Statistically significant (** P<0.005; ***P<0.001) values are indicated.

FIG. 9A-F. Molecular docking models of A. phagocytophilum OmpA-sLe^(x) interactions. (A) Predicted tertiary structure for A. phagocytophilum OmpA. The dotted line separates the regions encompassed by residues 19 to 74 and 75 to 205. Residues K60, G61, and K64 positions are indicated. (B) Stick representation of the N-terminal PSGL-1 amino acids 61 to 77 capped with sLe^(x) derived from PDB 1g1s. The sLe^(x) glycan extends off of threonine 73. sLe^(x) linkages and individual sugar residues are denoted. (C) Electrostatic surface map of A. phagocytophilum OmpA, as generated using the PyMol APBS plugin. The image is oriented as in (A). (D) The electrostatic surface map as in (C) rotated 180° around the y-axis. The dotted line is a demarcation between the regions encompassed by residues 19 to 74 and 75 to 205, which have overall cationic and anionic surface charges, respectively. (E and F) OmpA and sLe^(x) interactions predicted by the Autodock Vina algorithm. OmpA is presented as a ribbon model, sLe^(x) as a stick model, and hydrogen bonding by dotted lines. OmpA residue K64 is predicted to interact with α2,3-1080 sialic acid of sLe^(x) (E and F). Residue G61 is predicted to interact with either α2,3-sialic acid (E) or α1,3-fucose of sLe^(x) (F). Residue K60 is predicted to interact with β1,3 galactose of sLe^(x) (F).

FIG. 10A-B. OmpA is highly conserved among A. phagocytophilum isolates and its key binding residues exhibit variable conservation among Anaplasmataceae species. (A) Alignment of OmpA amino acid sequence from the A. phagocytophilum strain (isolated from a human patient in Massachusetts), with OmpA sequences from A. phagocytophilum strains HZ (human; New York), HGE1 (human; Minnesota), Dog (Minnesota), JM (jumping mouse; Minnesota), MRK (horse; California), ApVar-1 isolates CRT35 and CRT38 (both from ticks; Minnesota), and NorV2 (lamb; Norway). (B) Alignment of NCH-1 OmpA amino acids 19 to 74 with corresponding regions of OmpA homologs from the A. marginale St. Manes strain (AM854), A. marginale Florida strain (AMF640), A. marginale subsp. centrale Israel starin (ACIS00486) E. chaffeensis Arkansas strain (ECH0462), Ehrlichia canis Jake strain (Ecaj0563), and the Ehrlichia ruminantium Welgevonden strain (Erum5620). The binding domain corresponding to NCH-1 OmpA comprises residues 59 to 74. Numbers above the alignments in (A) and (B) denote amino acid position numbers. The arrows in (A) and (B) denote A. phagocytophilum OmpA G61 and K64, which were predicted to form interactions with sLe^(x) in FIG. 9 panels E and F and were shown to be critical for OmpA to bind to and mediate infection of mammalian host cells in FIGS. 8 and 11.

FIG. 11A-D. G61 and K64 are essential for recombinant OmpA to optimally bind to mammalian host cells and competitively inhibit A. phagocytophilum infection. GST1087 OmpA proteins having the CLNHL peptide inserted between OmpA amino acids 67 and 68 or having G61 and/or K64 mutated to alanine are unable to bind to competitively inhibit A. phagocytophilum infection of mammalian host cells. HL-60 cells were incubated with DC organisms in the presence of GST alone, GST-OmpA, GST-OmpA proteins bearing insertions of CLNHL (SEQ ID NO:75) between the indicated residues (A), or GST-OmpA proteins having the indicated amino acids substituted with alanine (B and C) for 1 h. After washing to remove unbound bacteria, host cells were incubated for 24 h and subsequently examined by immunofluorescence microscopy to determine the percentage of infected cells (A and B) or the mean number (±SD) of morulae per cell (C). Results shown in (A), (B), and (C) are the means+SD for six to twelve combined experiments. The data presented in panel C are the normalized values of six to twelve experiments. Statistically significant (** P<0.005; ***P<0.001) values are indicated. (D) Flow cytometric analysis of His-OmpA and His-OmpA proteins bearing alanine substitutions binding to RF/6A cells. Data are representative of two experiments with similar results.

FIG. 12A-C. Treatment with α1,3/4-fucosidase reduces A. phagocytophilum binding to PSGL-1 CHO cells and binding to and infection of RF/6A endothelial cells. PSGL-1 CHO cells (A) and RF/6A cells (B and C) were treated with α1,3/4-fucosidase (+ fucosidase) or vehicle control (− fucosidase). Fucosidase- and mock-treated cells were incubated with A. phagocytophilum DC organisms. Following the removal of unbound bacteria, the infection of RF/6A cells was allowed to proceed for 24 h prior to being assessed, while bacterial binding to PSGL-1 CHO and RF/6A cells was examined immediately. The mean±SD of bound DC bacteria per PSGL-1 CHO (A) or RF/6A cell (B) or percentage of infected RF/6A cells (C) were determined using immunofluorescence microscopy. Results shown are the means±SD for three combined experiments. Statistically significant (***P<0.001) values are indicated.

FIG. 13A-E. OmpA interacts with α1,3-fucose on mammalian host cell surfaces. (A to D) RF/6A cells were treated with α1,3/4-fucosidase (A and C), α2,3/6-sialidase (B and D), or vehicle control (− fucosidase and − sialidase, respectively). Glycosidase- and mock-treated cells were incubated with α1,3/6-fucose-specific lectin, AAL; the α2,3-sialic acid-specific lectin, MAL II; His-OmpA; or His-Asp14. (A) The host cells were fixed and screened using immmunofluorescence microscopy (A and B) or flow cytometry (C and D) to detect lectin, His-OmpA, or His-Asp14 binding to host cells. (E) AAL and MAL II competitively inhibit His-OmpA binding to mammalian host cells. RF/6A cells were incubated with AAL and MAL II, after which His-OmpA was added. Following the removal of unbound recombinant protein, His-OmpA bound on RF/6A cell surfaces was detected by flow cytometry. Statistically significant (***P<0.001) values are indicated. Results shown are representative of three experiments with similar results.

FIG. 14A-D. OmpA interacts with 6-sulfo sLe^(x) on RF/6A endothelial cell surfaces. (A) Schematic representations of sLe^(x) and 6-sulfo sLe^(x). Individual sugar and glycosidic linkages are indicated. (B and C) 6-sulfo sLe^(x) is present in high abundance relative to sLe^(x) on RF/6A cells. RF/6A cells were screened with sLe^(x)-specific antibodies, CSLEX1 and KM93; 6-sulfo sLe^(x)-specific antibody, G72; or IgM isotype control followed by detection of cell surface bound antibodies using immunofluorescence microscopy (B) and flow cytometry (C). (D) Antibody blocking of 6-sulfo sLe^(x) inhibits His-OmpA binding to RF/6A cells. RF/6A cells were incubated with CSLEX1, KM93, G72, IgM, or vehicle (Cells only) followed by the addition of His-OmpA, and washing to remove unbound recombinant protein. Flow cytometry was used to detect bound His-OmpA. Statistically significant (***P<0.001) values are indicated. Results shown are representative of two experiments with similar results.

FIG. 15A-D. OmpA coated beads bind to and are internalized by non-phagocytic endothelial cells. (A) Confirmation of recombinant OmpA conjugation to inert beads. Fluorescent OmpA-coated microspheres (OmpA beads) were incubated with OmpA antibody (αOmpA) or isotype control. Unconjugated (Control) beads were included as a negative control. Bound antibody was detected using immunofluorescence microscopy. (B to D) OmpA coated beads bind to and are internalized by nonphagocytic RF/6A cells. OmpA coated beads were incubated with RF/6A cells for 1 h after which unbound beads were washed off Cells were screened with OmpA antibody and examined using immunofluorescence (B and D) or scanning electron microscopy (C) to assess binding or were incubated further to allow for bead uptake. To assess for internalized beads, the host cells were treated with trypsin, washed, incubated overnight for the endothelial cells to re-adhere, fixed, and screened with OmpA antibody and immunofluorescence microscopy. (B) DIC, differential interference contrast microscopy. (C) Two scanning electron micrographs depicting bound or internalized OmpA coated beads. Arrows denote filopodia-like structures bound to beads. Scale bars are indicated. Results in (D) are representative of thirteen experiments with similar results. Statistically significant (** P<0.005; ****P<0.0001) values are indicated.

FIG. 16A-I. OmpA coated bead binding to and uptake by promyelocytic HL-60 cells involve OmpA residues G61 and K64 and are dependent on sLe^(x). (A) Scanning electron micrographs depicting OmpA coated beads bound to and being internalized by HL-60 cells. Arrows point to filopodia-like structures adhered to beads. Scale bars are indicated. (B and C) HL-60 cells were incubated with beads coated with OmpA, OmpA proteins having the indicated amino acids substituted with alanine, or non-coated control beads. The numbers of bound and internalized beads were determined using immunofluorescence microscopy. (D to I) HL-60 cells were incubated with α2,3/6-sialidase, α1,3/4-fucosidase, or vehicle only (D and E), sLe^(x) antibody CSLEX1 or IgM isotype control (F and G), or PSGL-1 N-terminus antibody KPL-1 or IgG isotype control (H and I) before being incubated with OmpA coated or non-coated control beads. The numbers of bound and internalized beads were assessed using immunofluorescence microscopy. Results in (B) through (I) are the mean±SD of triplicate samples and are representative of three independent experiments with similar results. Statistically significant (** P<0.005; ***P<0.001) values are indicated.

FIG. 17A-D. The Asp14 binding domain is contained within amino acids 113 to 124. (A) Pretreatment of A. phagocytophilum with Asp14113-124 antiserum inhibits infection of HL-60 cells in a dose-dependent manner. DC bacteria were incubated with 200 μg/ml of preimmune serum, 200 μg/ml of serum raised against full-length Asp14, or two-fold serially-diluted concentrations of anti-Asp1498-112 or anti-Asp14113-124 ranging from 0 to 200 μg/ml and then incubated with HL-60 cells. The infection was allowed to proceed for 24 h prior to being assessed by immunofluorescence microscopy for the percentage of infected cells. (B) A combination of antisera targeting OmpA59-74 and Asp14113-124 inhibits A. phagocytophilum infection of HL-60 cells better than serum targeting either binding domain alone. DC organisms were exposed to preimmune serum or antisera targeting OmpA59-74, Asp14113-124, OmpA59-74 plus Asp1498-112, or anti-Asp14113-124 together with OmpA59-74, OmpA23-40, or OmpA43-58 antibodies. The cells were fixed and screened using immunofluorescence microscopy to determine the percentages of infected cells. (C and D) OmpA59-74 and Asp14113-124 Fab fragments effectively inhibit A. phagocytophilum infection of HL-60 cells. DC bacteria were incubated with Fab fragments derived from preimmune serum, antibodies targeting OmpA23-40, OmpA41-58, OmpA59-74, Asp1498-112, Asp14113-124, or OmpA59-74 Fab fragment together with Asp14113-124 Fab fragment. The cells were fixed and screened to determine the percentages of infected cells (C) and morulae per cell (D). Results presented in (B) to (D) are relative to host cells that had been incubated with bacteria treated with preimmune serum. Results presented in (A) and (B) are the means±SD for three experiments. Results in (C) and (D) are the mean±SD of triplicate samples and are representative of two experiments with similar results. Statistically significant (* P<0.05; ** P<0.005; ***P<0.001) values are indicated.

FIG. 18A-C. A combination of antisera targeting the binding domains of OmpA, Asp14, and AipA blocks A. phagocytophilum infection of mammalian host cells. DC organisms were incubated with preimmune serum or antibodies specific for OmpA59-74 and Asp14113-124; or AipA9-21, AipA61-84, AipA165-182, or AipA183-201, either independently or in combination with OmpA59-74 and Asp14113-124 antibodies. Next, the bacteria were incubated with HL-60 cells. The infection was allowed to proceed for 24 h, after which the host cells were fixed and examined using immunofluorescence microscopy to determine the percentages of infected cells (A) and the number of morulae per cell (B). (C) To verify that the observed reductions in A. phagocytophilum infection were due to antisera mediated blocking of bacterial binding to HL-60 cell surfaces, the experiment was repeated except that DC organisms were incubated with antibodies targeting OmpA59-74 and/or Asp14113-124, and/or AipA9-21 prior to being incubated with host cells, and the numbers of bound bacteria per cell was assessed. Results presented are relative to host cells that had been incubated with bacteria treated with preimmune serum and are the means±SD for six combined experiments. Statistically significant (* P<0.05; ** P<0.005; ***P<0.001) values are indicated.

DETAILED DESCRIPTION

The detailed characterization of the AipA protein presented herein has led to the confirmation that it is an outer membrane protein and the discovery that it is in fact an invasin protein which is instrumental in host cell invasion by A. phagocytophilum bacterium. Further, the present disclosure establishes that AipA, and suitable fragments thereof which represent domains involved in bacterial invasion, can be used as both therapeutic agents (e.g. immunogenic agents and vaccinogens to prevent or treat infection); as diagnostic agents (e.g. as targets to detect antibodies to A. phagocytophilum in biological samples); and/or to generate antibodies which are used as therapeutic agents or as diagnostic agents to detect A. phagocytophilum AipA protein in biological samples. In some aspects, AipA and/or suitable effective fragments thereof are used for these purposes in combination with one or more other invasin proteins or suitable effective invasin fragments thereof, examples of which include OmpA and Asp14. Homologs of OmpA and Asp 14 may also be employed for these purposes.

The critical regions of OmpA and Asp14 that mediate infection are highly conserved among family members Aph, A. marginale, and closely related Ehrlichia species, such as E. chaffeensis, E. canis, and E. ruminatium, and may be highly conserved in A. platys. In particular, Aph and A. marginale are closely related and express many gene homologs, including Asp14 and OmpA and other surface antigens. The high degree of conservation makes these surface proteins ideal for producing a vaccine or immunogenic composition to provide protection from or therapy for multiple pathogens in humans and animals. AipA, however, has no known homologs in other Anaplasma and Ehrlichia species (all of which cause disease in humans and/or animals—cattle, horses, sheep, dogs, cats) or other non-Anaplasmataceae organisms. Thus, AipA is unique to A. phagocytophilum and is thus not employed alone to treat or diagnose infections other than A. phagocytophilum.

Accordingly, assays and methods of using the assays to distinguish exposure to or infection with A. phagocytophilum from exposure to or infection with a non-A. phagocytophilum Anaplasmataseae species are provided, e.g. to permit specialized treatment and/or to track the occurrence or frequency of A. phagocytophilum infections. A biological sample from a subject is thus screened with two or more of the agents described herein, at least one or which is AipA based or derived, and if a sample contains (is positive for the presence of) AipA proteins or antibodies to AipA proteins, it is concluded that the subject is infected with (or at least was previously exposed to) A. phagocytophilum. Previous exposure to A. phagocytophilum may result from a prior vaccination. Therefore, the methods of the invention may be used to determine if a subject has or has not been previously vaccinated. However, if the sample contains only OmpA and/or Asp14 proteins or antibodies, but AipA proteins/antibodies are absent, a conclusion is drawn that the subject is infected or has been exposed to an Anaplasmataseae species that is not A. phagocytophilum. If the subject is currently infected with A. phagocytophilum, then treatment specific for that organism may be undertaken, e.g. administration of an anti-A. phagocytophilum immune response eliciting composition (e.g. compositions comprising AipA protein or fragments thereof as described herein), administration of antibodies directed specifically to AipA protein or fragments thereof, etc.

In order to facilitate the understanding of the present invention, the following definitions are provided:

Aph: Anaplasma phagocytophilum or A. phagocytophilum, an Anaplasmataseae family bacterium that is tick-born and causes anaplasmosis in humans and animals.

Apl: Anaplasma platys or A. platys, an Anaplasmataseae family member bacterium that is tick-born and causes anaplasmosis that is restricted to dogs.

Anaplasmataceae: a family of closely related bacteria, including Anaplasma and Ehrlichia species. The genera Neorickettsia and Wolbachhia are also Anaplasmataceae, bacteria but do not cause anaplasmosis.

Antigen: term used historically to designate an entity that is bound by an antibody, and also to designate the entity that induces the production of the antibody. More current usage limits the meaning of antigen to that entity bound by an antibody, while the word “immunogen” is used for the entity that induces antibody production. Where an entity discussed herein is both immunogenic and antigenic, reference to it as either an immunogen or antigen will typically be made according to its intended utility. The terms “antigen”, “antigenic region” “immunogen” and “epitope” may be used interchangeably herein. As used herein, an antigen, immunogen or epitope is generally a portion of a protein (e.g. a peptide or polypeptide). AipA: 36.9-kilodalton Aph surface protein. AipA has no known homologs in other Anaplasma and Ehrlichia species or other non-Anaplasmataceae organisms. Asp14: 14-kilodalton Aph surface protein. Asp14 homologs are expressed by Anaplasmataceae family members, including Aph, A. marginale, Ehrlichia chaffeensis, E. canis, E. ewingii, and E. ruminatium. OmpA: Outer membrane protein A. OmpA homologs are expressed by Anaplasmataceae family members, including Aph, A. marginale, Ehrlichia chaffeensis, E. canis, E. ewingii, and E. ruminatium. DC and RC: Aph undergoes a biphasic developmental cycle, the kinetics of which have been tracked in promyelocytic HL-60 cells. The cycle begins with attachment and entry of an infectious dense-cored (DC) organism. Once intracellular, the DC differentiates to the non-infectious reticulate cell (RC) form and replicates by binary fission to produce a bacteria-filled organelle called a morula. Later, the RCs transition back to DCs, which initiate the next round of infection. Epitope: a specific chemical domain on an antigen that is recognized by a B-cell receptor, and which can be bound by secreted antibody. The term as used herein is interchangeable with “antigenic determinant”. An epitope may comprise a single, non-interrupted, contiguous chain of amino acids joined together by peptide bonds to form a peptide or polypeptide. Such an epitope can be described by its primary structure, i.e. the linear sequence of amino acids in the peptide chain. Epitope may also refer to conformational epitopes, which are comprised of at least some amino acids that are not part of an uninterrupted, linear sequence of amino acids, but which are brought into proximity to other residues in the epitope by secondary, tertiary and/or quaternary interactions of the protein. Residues in conformational epitopes may be located far from other residues in the epitope with respect to primary sequence, but may be spatially located near other residues in the conformational epitope due to protein folding.

Immunodominant epitope: The epitope on a molecule that induces the dominant, or most intense, immune response. The immunodominant epitope would elicit the greatest antibody titer during infection or immunization, as measured by, for example, the fraction of reactivity attributable to a certain antigen or epitope in an enzyme-linked immunosorbant assay as compared with the total responsiveness to an antigen set or entire protein.

Invasin domain: An invasin domain is a region of a pathogen's protein that binds a host cell and mediates intracellular signaling and pathogen entry into the host cell. In some cases, uptake of the pathogen results in the formation of a vacuole in which the intracellular pathogen will reside. The invasin domains of the invention are linear amino acid sequences within Asp14, OmpA, AipA or other surface proteins that are found on the outer membrane of the bacteria Aph and other Anaplasmataceae family members, and can vary slightly from one family member to the next. However, the invasin domain in each Asp14 homolog is critical for uptake of bacteria into host cells (known to be neutrophils and endothelial cells in the case of Anaplasmataceae). Invasin domains may be referred to herein as “effector domains”. Inclusive: with respect to linear amino acid sequence, inclusive refers to a sequence that includes the first- and last-numbered amino acids, e.g. “amino acids 1-87 inclusive” refers to a linear amino acid sequence that includes amino acids 1, 2, 3 . . . etc. up to and including amino acids . . . 85, 86 and 87 of a sequence, e.g. a sequence specified as a SEQ ID NO: or in referenced to a SEQ ID NO. Linker sequences: short peptide sequences encoding functional units that may be engineered or otherwise added at the ends or within recombinant proteins, polypeptides, peptides of interest. Linker sequences may be used as “handles” for protein purification, as detectable signals of expression or binding to other proteins or macromolecules, to modulate tertiary structure, or enhance antigenicity. Examples of linker sequences include, but are not limited to, an amino acid spacer, an amino acid linker, a signal sequence, a stop transfer sequence, a transmembrane domain, and a protein purification ligand. LINKER: a program to generate linker sequences for fusion proteins. Protein Engineering 13(5): 309-312, which is a reference that describes unstructured linkers. Structured (e.g. helical) sequence linkers may also be designed using, for example, existing sequences that are known to have that secondary structure, or using basic known biochemical principles to design the linkers. Tags: Recombinant protein sequences that can be added to the N- or C-terminus of a recombinant protein for the purpose of identification or for purifying the recombinant protein for subsequent uses. Examples of recombinant protein tags that may be useful in practicing the invention include, but are not limited to, glutathione-S-transferease (GST), poly-histidine, maltose binding protein (MBP), FLAG, V5, halo, myc, hemaglutinin (HA), S-tag, calmodulin, tag, streptavidin binding protein (SBP), Softag1™, Softag3™, Xpress tag, isopeptag, Spy Tag, biotin carboxyl carrier protein (BCCP), GFP, Nus-tag, strep-tag, thioredoxin tag, TC tag, and Ty tag. All such tags are well-known to those of ordinary skill in the art of recombinant protein production. Protein: Generally means a linear sequence of about 100 or more amino acids covalently joined by peptide bonds. Polypeptide: Generally means a linear sequence of about 55 to about 100 amino acids covalently joined by peptide bonds. Peptide: Generally means a linear sequence of about 55 or fewer amino acids covalently joined by peptide bonds. Note: The terms “peptide”, “polypeptide” and “protein” may be used interchangeably herein. Chimeric or fusion peptide or polypeptide: a recombinant or synthetic peptide or polypeptide whose primary sequence comprises two or more linear amino acid sequences which do not both occur in a single molecule in nature, or at least are not linearly contiguous in a single molecule in nature. (For example, they may both be present in a single molecule which comprises e.g. amino acids 1-15, but in the fusion or chimera, “amino acids 1-5” and amino acids 10-15″ are present but amino acids 6-9 are not present between the sequence represented by amino acids 1-5 and the sequence represented by amino acids 10-15.) The two or more sequences may be, for example, a peptide (e.g. an epitope or antigenic region) and a linker sequence, or two or more peptides (which may be the same or different) which are either contiguous or separated by a linker sequence, etc. Tandem repeats: two or more copies of nucleic acid or amino acid sequences encoding the same peptide, which are arranged in a linear molecule and are either contiguous or separated by a linker sequence, etc. Original or native or wild type sequence: The sequence of a peptide, polypeptide, protein or nucleic acid as found in nature. Recombinant peptide, polypeptide, protein or nucleic acid: peptide, polypeptide, protein or nucleic acid that has been produced and/or manipulated using molecular biology techniques such as cloning, polymerase chain reaction (PCR), etc. Synthetic peptide, polypeptide, protein or nucleic acid: peptide, polypeptide, protein or nucleic acid that has been produced using chemical synthesis procedures. Type-specific: associated primarily with a single phyletic group. Surface protein: A protein located on the outer surface membrane of a cell or bacterium.

Tables 1, 2 and 3 provide a list of the SEO ID NOS: for sequences described herein.

TABLE 1 Aph Sequence Listing with SEQ ID Numbers for Asp14, OmpA and AipA.. GENBANK ACCESSION SEQ ID PROTEIN # AND NO NAME NAME AMINO ACID SEQUENCE SEQ ID Full-length YP_504865 MIPLAPWKSISVVYMSGSDEYKEIIKQ NO: 01 Asp14 APH_0248 CIGSVKEVFGEGRFDDVVASIMKIVIQE KVLASSMQQDDTGTVGQIESGEGSGA RLSDEQVQQLMNSIREEFKDDLRAIKR RILKLERAVYGANTPKES SEQ ID Asp14 aa101- APH_0248 LRAIKRRILKLERAVYGANTPKES NO: 02 124 SEQ ID Asp14 aa113- APH_0248 RAVYGANTPKES NO: 03 124 SEQ ID Full length YP_504946 MLRRSSFFCLLALLSVTSCGTLLPDSN NO: 04 OmpA APH_0338 VGVGRHDLGSHRSVAFAKKVEKVYF DIGKYDLKGPGKKVILELVEQLRQDD SMYLVVIGHADATGTEEYSLALGEKR ANAVKQFIIGCDKSLAPRVTTQSRGK AEPEVLVYSTDAQEVEKANAQNRRA VIVVEFAHIPRSGVADMHAPVASSITS ENSNASAEGEDMEASEFSSATAN SEQ ID OmpA aa19-74 APH_0338 CGTLLPDSNVGVGRHDLGSHRSVAFA NO: 05 KKVEKVYFDIGKYDLKGPGKKVILEL VEQLR SEQ ID OmpA aa59-74 APH_0338 LKGPGKKVILELVEQL NO: 06 SEQ ID OmpA aa48-56 APH_0338 EKVYFDIGK NO: 07 SEQ ID OmpA APH_0338 GHADATGTEEYSLALG NO: 08 SEQ ID OmpA APH_0338 LVYSTDAQEVEKANAQNRRAV NO: 09 SEQ ID OmpA APH_0338 PDSNVGVGRHDLGSHRSVAFAKKVE NO: 10 KVYFDIGKYDLKGPGKKVILELVEQL RQDDSMYLVVIGHADATGTEEYSLAL GEKRANAVKQFIIGCDKSLAPRVTTQS RGKAEPEVLVYSTDAQEVEKANAQN RRAVIVVEFAHIPRSGVADM SEQ ID Asp14 aa101- APH_0248 LRAIKRRILKLE NO: 11 112 SEQ ID Asp14 aa19-60 APH_0248 DEYKEIIKQCIGSVKEVFGEGRFDDVV NO: 12 ASIMKMQEKVLASSM SEQ ID Full length APH_0915 MSFTMSKLSLDPTQGSHTAENIACSIF NO: 13 AipA DMVLGVKSTAKLLAGTWAGTSSTIW KTVTGAASSTKEASSKSYGTLRSSLGS SASRRMLGTCATAALCLTAPLLGAAA AGAAITCALITICMALLFLVLYTVLHI ASQMLRCASLLLSMVCNILHSTFTAT KSCLGGKSPARTTEERVAGDLDHKGV DSDRKHDAEKTEEKKHGLGSLCKSLA INLVSLMGTALVTTPIILLAVVLLVLVP VYLLCATVHHIYQGNYEDRNNDKGSS RGGGTTYYPMTMSASASEESLSSIISE GGLSKTSLPSYSAATATGTGNATGEV FSHSHSSGKSSSKPESRPESNLQNVVA ETMSQQQRSVS SEQ ID AipA aa9-21 APH_0915 SLDPTQGSHTAEN NO: 14 SEQ ID AipA aa1-87 APH_0915 MSFTMSKLSLDPTQGSHTAENIACSIF NO: 15 DMVLGVKSTAKLLAGTWAGTSSTIW KTVTGAASSTKEASSKSYGTLRSSLGS SASRRMLG

TABLE 2 Asp14 Homologs: Sequence Listing with SEQ ID Numbers SEQ ID Anaplasma AM936 MSGEDEYKEIIRQCIGSVKEVFGEGRFD NO: 16 marginale DVVASIMKNIQEKVLASSMKDGDPVG QIAADGVGNELYDRIADRLEERVSQKI SEDLRIIKKRLLRLERVVLGGGSVSGD AAAHQVSGNQPSQQNSSAAAEGG SEQ ID A. marginale AM936 LGGGSVSGDAAAHQVSGNQPSQQNSS NO: 17 AAAEGG SEQ ID A. marginale ACIS_00403 MSGEDEYKEIIRQCIGSVKEVFGEGRFD NO: 18 subspecies DVVASIMKMQEKVLASSMKDGDPVG Centrale QIAADGVGNELYDRIADRLEERVSQKI SEDLRIIKKRLLRLERVVLGGGSVSGD AAAAHQVSGNQPSQQNSSAAAEGG SEQ ID A. marginale ACIS_00403 LGGGSVSGDAAAAHQVSGNQPSQQNS NO: 19 subspecies SAAAEGG Centrale SEQ ID A. marginale & AM936 & MSGEDEYKEIIRQCIGSVKEVFGEGRFD NO: 20 A. marginale ACIS-00403 DVVASIMKMQEKVLASSM subspecies Centrale SEQ ID A. marginale & AM936 & DLRIIKKRLLRLERVV NO: 21 A. marginale ACIS-00403 subspecies Centrale SEQ ID Ehrlichia ECH_0377 MAEDDYKGVIKQYIDTVKEIVGDSKTF NO: 22 chaffeensis DQMFESVVRIQERVMAANAQNNEDG VIDNGDQVKRIGSSTSESISNTEYKELM EELKVIKKRILRLERKILKPKEEV SEQ ID E. chaffeensis ECH_0377 MAEDDYKGVIKQYIDTVKEIVGDSKTF NO: 23 DQMFESVVRIQERVM SEQ ID E. chaffeensis ECH_0377 ELKVIKKRILRLE NO: 24 SEQ ID E. chaffeensis ECH_0377 RKILKPKEEV NO: 25 SEQ ID E. canis Ecaj_0636 MADDEYKGVIQQYINTVKEIVSDSKTF NO: 26 DQMFESVVKIQERVMEANAQNDDGSQ VKRIGSSTSDSISDSQYKELIEELKVIKK RLLRLEHKVLKPKEGA SEQ ID E. canis Ecaj_0636 MADDEYKGVIQQYINTVKEIVSDSKTF NO: 27 DQMFESVVKIQERVM SEQ ID E. canis Ecaj_0636 ELKVIKKRLLRLE NO: 28 SEQ ID E. canis Ecaj_0636 HKVLKPKEGA NO: 29 SEQ ID E. ruminantium Erum6320 MADEDYKGVIKQYIDTVKEIVGDSKTF NO: 30 DQMFESVVKIQERVMAASAQNEANGA LVEGDSKMKRIRSADDSIAYTQSQELL EELKVLKKRIARLERHVFKSNKTEA SEQ ID E. ruminantium Erum6320 MADEDYKGVIKQYIDTVKEIVGDSKTF NO: 31 DQMFESVVKIQERVM SEQ ID E. ruminantittm Erum6320 ELKVLKKRIARLE NO: 32 SEQ ID E. ruminantium Erum6320 RHVFKSNKTEA NO: 33

TABLE 3 OmpA Homologs: Sequence Listing with SEQ ID Numbers SEQ ID Anaplasma AM854 MLHRWLALCFLASFAVTGCGLFSKEKV NO: 34 marginale GMDIVGVPFSAGRVEKVYFDFNKYEIKG SGKKVLLGLVERMKADKRSTLLIIGHTD SRGTEEYNLALGERRANAVKEFILGCDR SLSPRISTQSRGKAEPEVLVYSSDFKEAE KAHAQNRRVVLIVECQHSVSPKKKMAI KWPFSFGRSAAKQDDVGSSEVSDENPV DDSSEGIASEEAAPEEGVVSEEAAEEAPE VAQDSSAGVVAPE SEQ ID A. marginale AM854 LFSKEKVGMDIVGVPFSAGRVEKVYFDF NO: 35 NKYEIKGSGKKVLLGLVERMKADKRST LLII SEQ ID A. marginale ACIS_00486 MLHRWLALCLLASLAVTGCELFNKEKV NO: 36 subspecies NIDIGGVPLSAGRVEKVYFDFNKYEIKGS Centrale GKKVLLGLVERMKADKMSTLLIVGHTD SRGTEEYNLALGERRANAVKEFILGCDR SLSPRISTQSRGKAEPEILVYSSDFKEAEK AHAQNRRVVLIMECQHAASPKKARVSR WPFSFGRS SATQQDNGGGTVAAGSPGE DAPAEVVEPEETQEAGE SEQ ID A. marginale ACIS_00486 LFNKEKVNIDIGGVPLSAGRVEKVYFDF NO: 37 subspecies NKYEIKGSGKKVLLGLVERMKADKMST Centrale LLIV SEQ ID A. marginale & AM854 & AGRVEKVYFDFNKYEIKGSGKKVLLGL NO: 38 A. marginale ACIS-00486 VERMKAD subspecies Centrale SEQ ID A. marginale & AM936 & GHTDSRGTEEYNLALG NO: 39 A. marginale ACIS-00403 subspecies Centrale SEQ ID A. marginale & AM854 & RRANAVKEFILGCDRSLSPRISTQSRGKA NO: 40 A. marginale ACIS-00486 E subspecies Centrale SEQ ID A. marginale & AM854 & LVYSSDFKEAEKAHAQNRRVVLI NO: 41 A. marginale ACIS-00486 subspecies Centrale SEQ ID Ehrlichia ECH_0462 MKHKLVFIKFMLLCLILSSCKTTDHVPL NO: 42 chaffeensis VNVDHVFSNTKTIEKIYFGFGKATIEDSD KTILEKVMQKAEEYPDTNIIIVGHTDTRG TDEYNLELGKQRANAVKDFILERNKSLE DRIIIESKGKSEPAVLVYSNNPEEAEYAH TKNRRVVITLTDNLIYKAKSSDKDPSSN KTEQ SEQ ID Ehrlichia ECH_0462 NVDHVFSNTKTIEKIYFGFGKATIEDSDK NO: 43 chaffeensis TILEKVMQKAEEYPDTNIIIV SEQ ID Ehrlichia ECH_0462 IEDSDKTILEKVMQKAEEYPDTNIIIV NO: 44 chaffeensis SEQ ID Ehrlichia ECH_0462 GHTDTRGTDEYNLELGE NO: 45 chaffeensis SEQ ID Ehrlichia ECH_0462 QRANAVKDFILERNKSLEDRIIIESKGKS NO: 46 chaffeensis EPAV SEQ ID Ehrlichia ECH_0462 LVYSNNPEEAEYAHTKNRRVVI NO: 47 chaffeensis SEQ ID E. canis Ecaj_0563 MKHKLVFIKFILLCLILSSCKTTDHVPLV NO: 48 NTDHVFSNMKTIEKIYFDFGKATIGDSD KAILEKVIQKAQKDTNTNIVIVGHTDTR GTDEYNLELGEQRANAVKDFIIEHDKSL ENRITVQSKGKSEPAVINYSSNPEEAEH AHAKNRRVVITLTDNGNKTSQ SEQ ID E. canis Ecaj_0563 TTDHVPLVNTDHVFSNMKTIEKIYFDFG NO: 49 KATIGDSDKAILEKVIQKAQKDTNTNIVI V SEQ ID E. canis Ecaj_0563 GDSDKAILEKVIQKAQKDTNTNIVIV NO: 50 SEQ ID E. canis Ecaj_0563 GHTDTRGTDEYNLELGE NO: 51 SEQ ID E. canis Ecaj_0563 QRANAVKDFIIEHDKSLENRITVQSKGKS NO: 52 EPAV SEQ ID E. canis Ecaj_0563 LVYSSNPEEAEHAHAKNRRVVI NO: 53 SEQ ID E. ruminantium Erum5620 MRYQLIVANLILLCLTLNGCHFNSKHVP NO: 54 LVNVHNLFSNIKAIDKVYFDLDKTVIKD SDKVLLEKLVQKAQEDPTTDIIIVGHTDT RGTDEYNLALGEQRANAVRDFIISCDKS LEKRITVRSKGKSEPAILVYSNNPKEAED AHAKNRRVVITLVNNSTSTDNKVPTTTT PFNEEAHNTISKDQENNTQQQAKSDNIN NINTQQKLEQDNNNTPEVN SEQ ID E. ruminantium Erum5620 NSKHVPLVNVHNLFSNIKAIDKVYFDLD NO: 55 KTVIKDSDKVLLEKLVQKAQEDPTTDIII V SEQ ID E. ruminantium Erum5620 DSDKVLLEKLVQKAQEDPTTDIIIV NO: 56 SEQ ID E. ruminantium Erum5620 GHTDTRGTDEYNLALGE NO: 57 SEQ ID E. ruminantium Erum5620 QRANAVRDFIISCDKSLEKRITVRSKGKS NO: 58 EPAI SEQ ID E. ruminantium Erum5620 LVYSNNPKEAEDAHAKNRRVVI NO: 59

Aph surface proteins AipA, OmpA, and Asp14 have been identified as mediating bacteria-host cell binding and entry. Thus, the surface proteins AipA, OmpA, and Asp14 and fragments thereof that encompass an invasin domain, are used for diagnosing whether a patient has been suffering from Aph infection. Specifically, in one aspect, if antibodies to AipA are present in a suitable biological sample from a subject, then it can be concluded that the subject has been exposed to and/or is infected with Aph. In other aspects, if antibodies to AipA and one or both of OmpA and Asp14 are identified in a suitable biological sample from a subject, then it can be concluded that the subject has been exposed to and/or is infected with Aph. In some aspects, such determinations are made, e.g. by testing serum or other biological material from a subject suspected of an Aph infection by a suitable assay, such as an ELISA or immunoblot using, for example, antibodies to (e.g. specific antibodies to AipA (and optionally, to OmpA, and Asp14). Further, antibodies can also be raised against two or more of AipA, OmpA and Asp14, or to invasin domains from two or more of AipA, OmpA and Asp14, e.g. combined in a chimeric protein. If the antibodies bind to or interact with proteins or peptide fragments in the sample, then it can be determined that the subject has been exposed to, infected with, and/or is currently infected with Aph. Alternatively, if one or more of the amino acid sequences described herein (e.g. a form of the sequence that is labeled with a detectable label) is exposed to a biological sample, and it is found that there are antibodies in the sample which bind to the one or more amino acid sequences, then it can be concluded that the subject from whom the sample was taken has antibodies to Aph and thus has either been exposed to Aph bacteria and/or is still infected with Aph.

In other aspects, administration of AipA protein or suitable antigenic fragments thereof, or nucleic acids encoding the protein or fragments, is used to elicit an immunogenic response, e.g. a protective immunogenic response, in a subject. The immunogenic response may protect a naive subject from subsequent full-blown Aph infection when exposed to the bacterium (e.g. when bitten by an infected tick). Antibodies elicited by AipA administration bind to invasin proteins of bacteria that enter the bloodstream and prevent host cell invasion and establishment of a productive infection. Alternatively, administration of AipA protein or suitable antigenic fragments thereof, or nucleic acids encoding the protein or fragments, is used to provide treatment for an existing Aph infection by eliciting the production of antibodies to Aph. In some aspects, in addition to administration of AipA, one or both of OmpA and Asp14 proteins or suitable antigenic fragments thereof, or nucleic acids encoding such proteins and fragments, may be administered. For example, all three proteins, or suitable effective fragments thereof as described herein, may be administered.

In some aspects, what is provided herein is a polypeptide or protein comprising at least one “core” sequence or segment consisting of an invasin domain sequence from one or more of AipA, Asp14 and OmpA as described herein. In such constructs, which are generally chimeric or fusion polypeptides or proteins, the core sequence(s) is/are flanked on at least one of the amino terminus and carboxy terminus (i.e. at one or the other or both termini) by a sequence which is not found adjacent to the core sequence in nature. The sequence which is not found adjacent to the core sequence in nature may be a heterologous sequence (e.g. synthetic and/or from another species or strain) or may be a repeat or duplication of the same sequence. In some constructs, one core sequence is present alone; in other constructs, multiple core sequences of a single type (i.e. AipA, Asp14 or OmpA) are present. In yet other constructs, one core sequence from at least two of AipA, Asp14 and OmpA are present. In yet other constructions, multiple copies of one or more core sequence from at least two of AipA, Asp14 and OmpA are present.

It is contemplated that virtually any protein sequence, as well as its corresponding nucleic acid sequence coding for the protein sequence that is or includes SEQ ID NO: 14 may be used as described herein. This includes the full length sequence (e.g., SEQ ID NO:13) as well as any sequence of, for example from about 5-50 (or less than 5 or more than 50) amino acids before the beginning (amino terminus) or at the end (carboxy terminus) of the amino acid sequence of the invasin domain defined by SEQ ID NO:14. Such sequences include one or more amino acids or amino acid sequences which are adjacent to or which flank SEQ ID NO: 14 in nature (such as those which are present in SEQ ID NO:13). The polypeptide sequences as described herein may also be shortened on either the amino or carboxy terminus (or both) by one, two, or more amino acids to produce fragments within the context of the invention wherein the fragments produce the same or a similar protective effect. Alternatively, the polypeptide may be a chimera or fusion protein which comprises flanking amino acids sequences which are not adjacent to the native sequence in nature. For example, the adjacent sequences may be corresponding amino acids which are from different but related species (e.g. other Anaplasmataceae); or amino acids which are from different species (e.g. from other bacteria or eukaryotes of interest, e.g. from infectious agents); or from a synthetic sequence, e.g. various tags such as histidine or glutathione S-transferase (GST) tags, linkers, spacers, targeting sequences, etc. as described elsewhere herein).

Further, a single recombinant polypeptide may comprise one copy, or more than one copy (a plurality, two or more) of each of these different sequences, all of which include SEQ ID NO: 14. A single polypeptide may contain two or more copies of a single sequence, a single copy of one sequence and two or more copies of one or more different sequences, or two or more copies of at least two different sequences. In exemplary embodiments, a polypeptide of the invention includes one or more copies of SEQ ID NO:14 and optionally one or more copies of at least one of SEQ ID NO:03 and SEQ ID NO:06 on a single polypeptide or multiple polypeptides. Another aspect of the disclosure provides a mixture of at least two of any of the peptides and/or polypeptides described herein.

In some embodiments, the composition consists of or comprises three polypeptides with amino acid sequences encoding an AipA, OmpA, or Asp14 invasin domain. The Asp14 invasin domain lies within aa113-124 (SEQ ID NO:03). In other embodiments, polypeptide fragments such as Asp14 aa101-124 (SEQ ID NO:02) or the full length protein (SEQ ID NO:01) are used. In another embodiment, the composition of the invention comprises the invasin domain of OmpA, which lies within aa59-74 (SEQ ID NO:04). In other embodiments, a larger fragment of OmpA encompassing aa19-74 (SEQ ID NO:05), or the full length OmpA protein (SEQ ID NO:06) is used.

There is currently no means for preventing transmission of the bacteria causing anaplasmosis or HGA. While antibiotic treatments exist, these treatments are not advised for some groups of patients. In one embodiment, the invention is a vaccine for prevention or treatment of anaplasmosis and HGA. One embodiment of the invention is a pharmaceutically acceptable composition comprising one or a plurality of any one of or a mixture of at least two amino acid sequences which are or include the amino acid sequences which are identified as SEQ ID NO:01, 02, 03, 04, 05, 06, 13, 14, or 15. Administration of the composition of the invention stimulates an immune response in a subject and production of antibodies against AipA, Asp14, OmpA, or all three. Because AipA, OmpA, and Asp14 are on the outer surface of Aph bacteria, antibodies produced by the subject will block binding of bacteria to host cells and interfere with uptake into vacuoles. Bacteria unable to enter host cells are detected by the host immune system and cleared from the body. Blockade can occur at the point of entry into neutrophils or endothelial cells or transfer between these two host cell types. Interruption of the zoonotic life cycle provides a further benefit to public health and well-being by breaking the chain of disease transmission to others.

Aside from commercial assays to detect Apl in dogs, there is no specific assay to rapidly confirm Anaplasmataceae infection, or accurately diagnose HGA or anaplasmosis. In one embodiment, the invention provides a method to detect the presence of Aph AipA, OmpA, and/or Asp14 in assays of biological samples obtained from subjects to bind to antibodies produced by an Anaplasmataceae-infected individual, either of which would be diagnostic for HGA or anaplasmosis. The preferred composition for diagnostic testing may comprise full length AipA (SEQ ID NO:13), Asp14 (SEQ ID NO: 3), and/or OmpA (SEQ ID NO:06). However, compositions comprising fragments of AipA such as SEQ ID NO:14 or a mixture of at least two of SEQ ID NO:13, 14, and 15 are also contemplated. Likewise, a composition comprising fragments of OmpA, such as SEQ ID NO:04 and/or 05, and any mixtures of at least two of SEQ ID NO:04, 05, and 06 are also contemplated. Fragments of Asp14, such as SEQ ID NO:01 and/or 02, are also contemplated, as are any mixtures of at least two of SEQ ID NO:01, 02, and 03. The assay used to detect antibodies may be any type of immunoassay, such as an immunoblot or an enzyme-linked immunosorbent assay. The test sample may be any type of body fluid, such as blood, plasma, serum, urine, saliva, or other body fluid. Tissues or cells may also be used, such as tissue sections or cell preparations adhered to slides or coverslips for immunohistochemical staining. In an exemplary embodiment, the assay used is an ELISA with each protein type to independently detect antibodies to AipA, Asp14, and OmpA, however, a combination to detect AipA, Asp14, and OmpA antibodies in one ELISA is also contemplated.

In addition to sequences for OmpA, Asp14, and AipA shown in Table 1, and homologs shown in Tables 2-3, other surface proteins that Aph preferentially expresses in human versus tick cells may be used. Table 4 shows examples of proteins that can be included in the “cocktail” of peptides, polypeptides or protein sequences of the composition of the invention. Examples of these include APH_1325 (Msp2), APH_1235, APH_1378, APH_1412, APH_0346, APH_0838, APH_0839, APH_0874, and APH_0906 because all are upregulated 3- to 60-fold during RC-DC transition, DC exit, and/or reinfection and our surface proteomic study indicates that they are surface proteins. The file names for each of the aforementioned proteins are from the A. phagocytophilum HZ annotated genome. A similar expression profile is exhibited by APH_1235, which is another late stage gene that is upregulated 70-fold (Troese et al., 2011). Studies by Mastronunzio and colleagues suggest that APH_1235 is an A. phagocytophilum surface protein. P44 is a 44 kilodalton surface protein and is the bacterium's major surface protein. Synonyms of P44 are Msp2 (major surface protein 2) and Msp2 (P44). All Anaplasma species encode P44 proteins and there are huge repertoires of P44 genes in these bacterial species' chromosomes. For instance, the annotated Aph strain HZ genome encodes 113 P44 proteins. These exist as complete genes or pseudogenes (incomplete genes). There is one expression site for p44 genes. Basically, different p44 genes get shuffled into the expression site by a process known as gene conversion with the end result being that Aph (and other Anaplasma species) can vary the P44 protein on their cell surfaces, a process called antigenic variation. This enables them to perpetually evade the humoral immune response.

TABLE 4 Anaplamatacaea Surface Proteins Sequence Listing and SEQ ID Numbers SEQ ID NO: 60 Full-length APH_1378 Genbank Accession No: YP_505877 SEQ ID NO: 61 Full-length APH_1412 Genbank Accession No: YP_505903 SEQ ID NO: 62 Full-length APH_0346 Genbank Accession No: YP_504953 SEQ ID NO: 63 Full-length APH_0838 Genbank Accession No: YP_505415 SEQ ID NO: 64 Full-length APH_0839 Genbank Accession No: YP_505416 SEQ ID NO: 65 Full-length APH_0874 Genbank Accession No: YP_505450 SEQ ID NO: 66 Full-length APH_0906 Genbank Accession No: YP_505479 SEQ ID NO: 67 Full-length APH_1325 Genbank Accession (Msp2) No: YP_505833 SEQ ID NO: 68 Full-length APH_1235 Genbank Accession No: YP_505764

In addition to polypeptides sequences from Aph surface proteins, other sequences may be included in the polypeptides of the invention. Such sequences include but are not limited to antigenic peptide sequences such as linker sequences which in and of themselves are antigenic. Examples of recombinant protein tags that may be useful in practicing the invention include but are not limited to glutathione-S-transferease (GST), poly-histidine, maltose binding protein (MBP), FLAG, V5, halo, myc, hemaglutinin (HA), S-tag, calmodulin, tag, streptavidin binding protein (SBP), Softag1™, Softag3™, Xpress tag, isopeptag, Spy Tag, biotin carboxyl carrier protein (BCCP), GFP, Nus-tag, strep-tag, thioredoxin tag, TC tag, and Ty tag. Examples of linker sequences include but are not limited to an amino acid spacer, an amino acid linker, a signal sequence, a stop transfer sequence, a transmembrane domain, and a protein purification ligand. It should also be recognized that a multitude of other such sequences are known to those of skill in the art, and inclusion of other antigenic, linker, or tag sequences is contemplated.

Those of skill in the art will recognize that, while in some embodiments of the invention, the amino acid sequences that are chosen for inclusion in the polypeptides of the invention correspond exactly to the primary amino acid sequence of the original or native sequences of an AipA, Asp14, or OmpA protein, this need not always be the case. The amino acid sequence of an epitope that is included in the polypeptides of the invention may be altered somewhat and still be suitable for use in the present invention. For example, certain conservative amino acid substitutions may be made without having a deleterious effect on the ability of the polypeptides to elicit an immune response. Those of skill in the art will recognize the nature of such conservative substitutions, for example, substitution of a positively charged amino acid for another positively charged amino acid (e.g. K for R or vice versa); substitution of a negatively charged amino acid for another negatively charged amino acid (e.g. D for E or vice versa); substitution of a hydrophobic amino acid for another hydrophobic amino acid (e.g. substitution of A, V, L, I, W, etc. for one another); etc. All such substitutions or alterations of the sequences of the polypeptides that are disclosed herein are intended to be encompassed by the present invention, so long as the resulting polypeptides still function to elicit a suitable immune response. In addition, the amino acid sequences that are included in the polypeptides or any chimeric proteins of the invention need not encompass a full length native polypeptide. Those of skill in the art will recognize that truncated versions of amino acid sequences that are known to be or to contain antigenic polypeptides may, for a variety of reasons, be preferable for use in the practice of the invention, so long as the criteria set forth for an epitope is fulfilled by the sequence. Amino acid sequences that are so substituted or otherwise altered may be referred to herein as “based on” or “derived from” the original wild type or native sequence. In general, the AipA, Asp14, or OmpA proteins or polypeptide fragments from which the linear epitopes are “derived” or on which the linear epitopes are “based” are the AipA, Asp14, or OmpA proteins or peptide fragments as they occur in nature. These natural AipA, Asp14, or OmpA proteins may alternatively be referred to as native or wild type proteins.

Such changes to the primary sequence may be introduced for any of a variety of reasons, for example, to eliminate or introduce a protease cleavage site, to increase or decrease solubility, to promote or discourage intra- or inter-molecular interactions such as folding, ionic interactions, salt bridges, etc, which might otherwise interfere with the presentation and accessibility of the individual epitopes along the length of a peptide or polypeptide. All such changes are intended to be encompassed by the present invention, so long as the resulting amino acid sequence functions to elicit a protective antibody response in a host to whom it is administered. In general, such substituted sequences will be at least about 50% identical to the corresponding sequence in the native protein, preferably about 60 to 70, or even 70 to 80, or 80 to 90% identical to the wild type sequence, and preferably at least about 95, 96, 97, 98, 99, or even 100% identical to a native AipA, Asp14, or OmpA sequence or peptide fragment. The reference native AipA, Asp14, or OmpA sequence or peptide fragment may be from any suitable type of Anaplasmataceae, e.g. from any Anaplasmataceae which is known to infect mammals.

In some embodiments of the invention, individual linear epitopes in a chimeric vaccinogen are separated from one another by intervening sequences that are more or less neutral in character, i.e. they do not in and of themselves elicit an immune response to Anaplasmataceae. Such sequences may or may not be present between the epitopes of a chimera. If present, they may, for example, serve to separate the epitopes and contribute to the steric isolation of the epitopes from each other. Alternatively, such sequences may be simply artifacts of recombinant processing procedures, e.g. cloning procedures. Such sequences are typically known as linker or spacer peptides, many examples of which are known to those of skill in the art. See, for example, Crasto, C. J. and J. A. Feng. 2000.

In addition, other elements may be present in chimeric proteins, for example leader sequences or sequences that “tag” the protein to facilitate purification or detection of the protein, examples of which include but are not limited to tags that facilitate detection or purification (e.g. S-tag, Flag-tag, Histidine tags), other antigenic amino acid sequences such as known T-cell epitope containing sequences and protein stabilizing motifs, etc. In addition, the chimeric proteins may be chemically modified, e.g. by amidation, sulfonylation, lipidation, or other techniques that are known to those of skill in the art.

The invention further provides nucleic acid sequences that encode the proteins, protein fragments, polypeptides, peptides and/or chimeric proteins described herein. Such nucleic acids include DNA, cDNA, RNA (e.g. mRNA), and/or hybrids thereof, and the like, both single and double stranded, and complements thereof. Further, the invention comprehends vectors which contain or house one or more of such coding sequences, or sequences complementary to the coding sequences. Examples of suitable vectors include but are not limited to plasmids, cosmids, viral based vectors, expression vectors, etc. In some embodiments, the vector is a plasmid expression vector. In other aspects, the vector is a vector suitable for administration to a mammal such as a human, e.g. an attenuated viral vector such as an adenoviral, retroviral, herpes simplex, or other viral vector which is replication competent. In other aspects, the vector is a host cell harboring the nucleic acid. The encoding sequences are typically configured within the vectors so as to be translatable, e.g. by being operationally connected to a functional promoter sequence or region. The vectors may or may not be capable of replication, but are capable of being translated to produce the encoded polypeptide(s).

Nucleic acids encoding the sequences of interest may also be present in or associated with, for example, lipoplexes, polymersomes, polyplexes, dendrimers, organic and inorganic nanoparticles, etc.

The invention also provides host cells containing the nucleic acids of the invention. The host cell can be any cell that can grow in culture, be transformed or transfected with heterologous nucleotide sequences and can express those sequences. These include bacteria, such as E. coli, Bacillus, yeast and other fungi, plant cells, insect cells, and mammalian cells. In addition, expression of these sequences in higher eukaryotic host cells may be transient or stable.

The chimeric proteins of the invention may be produced by any suitable method, many of which are known to those of skill in the art. For example, they may be chemically synthesized, or produced using recombinant DNA technology (e.g. in bacterial cells, in cell culture (mammalian, yeast or insect cells), in plants or plant cells, or by cell-free prokaryotic or eukaryotic-based expression systems, by other in vitro systems, etc.). In some embodiments, the polypeptides are produced using chemical synthesis methods.

The present invention also provides compositions for use in eliciting an immune response. The compositions comprise one or more peptides, polypeptides, or proteins as described herein, or nucleic acids encoding them (e.g. as the nucleic acid per se, or in a vector such as a host cell comprising the nucleic acid). The compositions may be utilized as immunogenic composition and/or vaccines to prevent or treat anaplasmosis, particularly when manifested in humans as HGA. By eliciting an immune response, it is meant that administration of the antigen causes the synthesis of specific antibodies (at a titer as described above) and/or cellular proliferation, as measured, e.g. by ³H thymidine incorporation, or by other known techniques. By “vaccine” we mean a linear polypeptide, a mixture of linear polypeptides or a chimeric or fusion polypeptide that elicits an immune response, which results in protection of an organism against challenge with an Anaplasmataceae species bacterium. The protective response either wholly or partially prevents or arrests the development of symptoms related to anaplasmosis or HGA infection (i.e. the symptoms of anaplasmosis), in comparison to a non-vaccinated (e.g. adjunct alone) control organisms, in which disease progression is not prevented. The compositions include one or more isolated and substantially purified polypeptides or chimeric peptides as described herein, and a pharmacologically suitable carrier. The polypeptides or chimeric peptides in the composition may be the same or different, i.e. the composition may be a “cocktail” of different polypeptides or chimeric peptides, or a composition containing only a single type of polypeptide or chimeric peptide. The preparation of such compositions for use as vaccines is well known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions, however solid forms such as tablets, pills, powders and the like are also contemplated. The liquid may be an aqueous liquid. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared. The preparation may also be emulsified. The active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. The vaccine preparations of the present invention may further comprise an adjuvant, suitable examples of which include but are not limited to Seppic, Quil A, Alhydrogel, etc. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added. The composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of polypeptides or chimeric peptides in the formulations may vary. However, in general, the amount in the formulations will be from about 0.01-99%, weight/volume.

The methods involve administering a composition comprising proteins, protein fragments (peptides), recombinant polypeptides and/or chimeric peptides described herein, or by administering nucleic acids encoding these proteins, fragments and recombinant polypeptides and chimeric peptides, in a pharmacologically acceptable carrier to a mammal. The nucleic acids may be housed in a vector (e.g. a host cell) as described herein. The mammal may be a human, but this need not always be the case. Because anaplasmosis is a zoonotic disease that causes anaplasmosis in all known mammalian hosts, veterinary applications of this technology are also contemplated. The vaccine preparations of the present invention may be administered by any of the many suitable means which are well known to those of skill in the art, including but not limited to by injection, inhalation, orally, intranasally, by ingestion of a food product containing the polypeptides or chimeric peptides, etc. In some embodiments, the mode of administration is subcutaneous or intramuscular. In addition, the compositions may be administered in conjunction with other treatment modalities such as substances that boost the immune system, various anti-bacterial chemotherapeutic agents, antibiotics, and the like.

The present invention provides methods to elicit an immune response to Anaplasmataceae and/or to vaccinate against Anaplasmataceae infection in mammals. In one embodiment, the mammal is a human. However, those of skill in the art will recognize that other mammals exist for which such vaccinations would also be desirable, e.g. the preparations may also be used for veterinary purposes. Examples include but are not limited to companion “pets” such as dogs, cats, etc.; food source, work and recreational animals such as cattle, horses, oxen, sheep, pigs, goats, and the like; or wild animals that serve as a reservoir of Anaplasmataceae, particularly wild animals adapted to living in close proximity to urban areas (e.g. mice, deer, rats, raccoons, opossum, coyotes, etc). The compositions of the invention may be used as bait vaccines, such as bait containing any of the vaccine preps described herein (e.g. mouse bait, deer bait, etc.)

Also, animals in zoos and reserves may benefit from protection against Anaplasmataceae infection.

The invention also provides a diagnostic and a method for using the diagnostic to identify individuals who have antibodies to the epitopes contained within the polypeptides or chimeric proteins of the invention. A biological sample from an individual (e.g. a human, a deer, or other mammals susceptible to infection by Anaplasmataceae) suspected of having been exposed to Anaplasmataceae, or at risk for being exposed to Anaplasmataceae, is contacted with the peptides, polypeptides, or chimeric proteins of the invention. Using known methodology, the presence or absence of a binding reaction between the polypeptides or chimeric proteins and antibodies in the biological sample is detected. A positive result (i.e. binding occurs, thus antibodies are present) indicates that the individual has been exposed to and has previously mounted an immune response to, and/or is infected with Anaplasmataceae. Further, the diagnostic aspects of the invention are not confined to clinical use or home use, but may also be valuable for use in the laboratory as a research tool, e.g. to identify Anaplasmataceae bacteria isolated from ticks, to investigate the geographical distribution of Anaplasmataceae species and strains, etc.

The present invention also encompasses antibodies to the epitopes and/or to the polypeptides or chimeric proteins disclosed herein. Such antibodies may be polyclonal, monoclonal, or chimeric, and may be generated in any manner known to those of skill in the art. In a preferred embodiment of the invention, the antibodies are bactericidal, i.e. exposure of Anaplasmataceae bacteria to the antibodies causes death of the bacteria. Such antibodies may be used in a variety of ways, e.g. as detection reagents to diagnose prior exposure to Anaplasmataceae, as a reagent in a kit for the investigation of Anaplasmataceae, to treat Anaplasmataceae infections, etc.

Alternatively, appropriate antigen fragments or antigenic sequences or epitopes may be identified by their ability, when included in polypeptides or chimeric proteins, to elicit suitable antibody production to the epitope in a host to which the polypeptides or chimeric proteins are administered. Those of skill in the art will recognize that definitions of antibody titer may vary. Herein, “titer” is taken to be the inverse dilution of antiserum that will bind one half of the available binding sites on an ELISA well coated with 100 ng of test protein. In general, suitable antibody production is characterized by an antibody titer in the range of from about 100 to about 100,000, and preferably in the range of from about 10,000 to about 10,000,000. Alternatively, and particularly in diagnostic assays, the “titer” should be about three times the background level of binding. For example, to be considered “positive”, reactivity in a test should be at least three times greater than reactivity detected in serum from uninfected individuals. Preferably, the antibody response is protective, i.e. prevents or lessens the development of symptoms of disease in a vaccinated host that is later exposed to Anaplasmataceae, compared to an unvaccinated host.

Before exemplary embodiments of the present invention are described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention.

Example 1. Anaplasma phagocytophilum Surface Protein AipA Mediates Invasion of Mammalian Host Cells

Summary

Anaplasma phagocytophilum, which causes granulocytic anaplasmosis in humans and animals, is a tick-transmitted obligate intracellular bacterium that mediates its own uptake into neutrophils and non-phagocytic cells. Invasins of obligate intracellular pathogens are attractive targets for protecting against or curing infection because blocking the internalization step prevents survival of these organisms. The complement of A. phagocytophilum invasins is incompletely defined. Here, the significance of a novel A. phagocytophilum invasin protein, AipA is reported. A. phagocytophilum induced aipA expression during transmission feeding of infected ticks on mice. The bacterium upregulated aipA transcription when it transitioned from its non-infectious reticulate cell morphotype to its infectious dense-cored morphotype during infection of HL-60 cells. AipA localized to the bacterial surface and was expressed during in vivo infection. Of the AipA regions predicted to be surface-exposed, only residues 1 to 87 (AipA₁₋₈₇) were found to be essential for host cell invasion. Recombinant AipA₁₋₈₇ protein bound to and competitively inhibited A. phagocytophilum infection of mammalian cells. Antiserum specific for AipA₁₋₈₇, but not other AipA regions, antagonized infection. Additional blocking experiments using peptide-specific antisera narrowed down the AipA invasion domain to residues 9 to 21. An antisera combination targeting AipA₁₋₈₇ together with two other A. phagocytophilum invasins, OmpA and Asp14, nearly abolished infection of host cells. This study identifies AipA as an A. phagocytophilum surface protein that is critical for infection, demarcates its invasion domain, and establishes a rationale for targeting multiple invasins to protect against granulocytic anaplasmosis.

Introduction

Anaplasma phagocytophilum is an obligate intracellular bacterium in the order Rickettsiales and family Anaplasmataceae that infects neutrophils to cause granulocytic anaplasmosis in humans and animals. Though primarily an Ixodes spp. tick-borne illness (Truchan et al., 2013), human granulocytic anaplasmosis (HGA) can also be transmitted perinatally, nosocomially, and through blood transfusion (Annen et al., 2012; Carlyon, 2012; Jereb et al., 2012; Alhumaidan et al., 2013). The disease presents as a non-specific febrile illness that can be accompanied by leukopenia, thrombocytopenia, elevated levels of serum transaminases, and increased susceptibility to potentially fatal secondary infections (Truchan et al., 2013). HGA is an emerging infection in the United States, Europe, and Asia (Truchan et al., 2013). The number of reported HGA cases in the United States increased over six-fold from 2003 to 2012, the latest period for which disease reporting statistics are available (Hopkins et al., 2005; Centers for Disease Control and Prevention, 2013).

A. phagocytophilum undergoes a biphasic developmental cycle that begins when an infectious dense-cored (DC) organism binds to and enters its host cell, where it resides within a host cell-derived vacuole. Between 4 and 8 h, the DC develops into the non-infectious reticulate cell (RC) morphotype that subsequently divides by binary fission to yield a bacteria-filled vacuolar inclusion. From 8 to 20 h, the intravacuolar population consists exclusively of replicating RCs. Most RCs transition back into DCs between 28 and 32 h. DCs then exit host cells between 28 to 36 h and initiate the next round of infection (Troese et al., 2009). A. phagocytophilum OMPs that are upregulated during RC-to-DC transition, bacterial exit, and reinfection are attractive targets to evaluate for both their roles in infection and their prospect as protective antigens.

Given the potential severity of HGA, the limited choices of antibiotics for treating the disease, and the lack of a vaccine, a thorough understanding of A. phagocytophilum cellular invasion is critical. OmpA (APH0338) and Asp14 (14-kDa A. phagocytophilum surface protein; APH0248) were recently identified as being important for A. phagocytophilum entry into mammalian cells (Ojogun et al., 2012; Kahlon et al., 2013). OmpA binds to α2,3-sialic acid of the sialyl Lewis x (sLe^(x)) tetrasaccharide that caps P-selectin glycoprotein ligand-1 (PSGL-1) on myeloid cell surfaces. OmpA also recognizes α2,3-sialic acid residues that decorate glycoproteins on endothelial cells (Ojogun et al, 2012). The Asp14 receptor is unknown. Evidence implicates involvement of one or more A. phagocytophilum invasins in addition to OmpA and Asp14 in mediating infection (Kahlon et al., 2013). Identifying these invasins and evaluating if targeting them alone or in concert with OmpA and Asp14 can block bacterial entry will lead to the development of effective prophylaxes against HGA.

A whole genome transcriptional profiling study revealed A. phagocytophilum genes that are upregulated during infection of mammalian versus tick cells (Nelson et al., 2008). Several of these encode putative OMPs, one of which is APH0915. In this study, it is shown that APH0915, hereafter referred to as AipA (A. phagocytophilum invasion protein A), is important for bacterial entry into mammalian cells and identify its invasion domain. It is further demonstrated that a combination of antisera targeting AipA, OmpA, and Asp14 synergistically blocks infection. These findings not only advance understanding of how A. phagocytophilum employs multiple invasins to promote infection, but also results in the development of a multi-target vaccine that protects against granulocytic anaplasmosis.

Materials and Methods

Cultivation of Uninfected and A. phagocytophilum-Infected Host Cell Lines

Human promyelocytic HL-60 cells (CCL-240; American Type Culture Collections (ATCC), Manassas, Va.), RF/6A (rhesus monkey choroidal endothelial cells, ATCC CRL-1780), ISE6 cells and A. phagocytophilum (NCH-1 strain) infected HL-60, RF/6A, or ISE6 cells were cultured as previously described (Huang et al., 2012). PSGL-1 CHO cells and untransfected CHO cells were maintained as previously described (Troese et al., 2009).

In Silico Analyses of AipA

The AipA sequence was assessed for transmembrane domains using the TMpred and TMHMM algorithms (Hofmann et al., 1993; Krogh et al., 2001), each of which yielded highly similar predictions. Results obtained using TMpred are presented in FIG. 1. Protean, which is part of the Lasergene® software package (version 8.02; DNASTAR®, Madison, Wis.), was used to assess AipA for regions of hydrophobicity and probability of being surface-exposed using the Kyte-Doolittle (Kyte et al., 1982) and Emini (Emini et al., 1985) algorithms, respectively.

Recombinant Protein and Antiserum Production

A PCR amplicon of aipA (aph0915) nucleotides 1 to 261, encoding AipA amino acids 1 to 87, was generated using primers 5′-CACCTTGAGTTTTACAATGTCGAAGTTATCGC-3′ (SEQ ID NO:69; the first four nucleotides correspond to a Gateway entry vector-compatible sequence) and 5′-CTATCCTAGCATCCTTCTAGAAGCGGAAG-3′ (SEQ ID NO:70; the first three nucleotides denote an added stop codon). A PCR product corresponding to aipA nucleotides 745 to 1068, encoding AipA amino acids 249 to 355, was generated using primers 5′-CACCATCTATCAAGGAAATTACGAAGATCGCAAC-3′ (SEQ ID NO:71) and 5′-GAGCAGCATGCTTTA-3′ (SEQ ID NO:72). The amplicons were cloned into the pDest-15 vector (Life Technologies, Carlsbad, Calif.) downstream of and in frame with the gene encoding GST as described previously (Ojogun et al., 2012). Expression and purification GST-tagged AipA residues 1 to 87 (GST-AipA₁₋₈₇), 249 to 355 (GST-AipA₂₄₉₋₃₅₅), and GST alone were performed as previously described (Troese et al., 2011). GST-tagged full-length AipA and AipA amino acids 165 to 204 remained insoluble over a wide range of conditions and thus could not be purified. Generation of murine polyclonal antisera against each GST fusion protein was performed as described previously (Troese et al., 2011). Rabbit polyclonal antisera were raised against synthetic keyhole limpet hemocyanin (KLH)-conjugated peptides corresponding to AipA amino acid residues 9 to 21, 61 to 84, 165 to 182, and 185 to 201 (New England Peptide, Gardner, Mass.). Specificity of each AipA peptide antiserum for its target peptides was determined by the enzyme-linked immunosorbent assay using the TMB substrate kit (Thermo Scientific, Waltham, Mass.) according to the manufacturer's instructions.

Differential aipA Expression Studies

HL-60 cells were synchronously infected with A. phagocytophilum DC organisms (Troese et al., 2009). The infection time course was allowed to proceed for 36 h, a time period that enabled the bacteria to complete their biphasic developmental cycle and initiate a second round of infection (Troese et al., 2009). RNA isolated from aliquots taken every 4 hours was subjected to reverse transcriptase-quantitative PCR (RT-qPCR) as described previously (Troese et al., 2011) using AipA specific primers 5′-CCTCAACTAAAGAAGCGTCATCAAA-3′ (SEQ ID NO:73) and 5′-GTACGGTGTACAAAACGAGGAACA-3′ (SEQ ID NO:74), which targeted nucleotides 179 to 388. Relative aipA transcript levels were normalized to the transcript levels of the A. phagocytophilum 16s rRNA gene (aph1000) using the 2^(−ΔΔCT) method (Livak et al., 2001; Kahlon et al., 2013). To determine if aipA was transcriptionally upregulated in the DC versus RC morphotype, normalized aipA transcript levels were calculated as fold changes in expression relative to expression at 16 h, a time point at which the entire A. phagocytophilum population existed in the RC form (Troese et al., 2009; Mastronunzio et al., 2012). aipA expression during blood meal acquisition by A. phagocytophilum infected nymphs from mice was monitored as described (Mastronunzio et al., 2012) using AipA primers targeting nucleotides 179 to 388. As a control, expression of the A. phagocytophilum groEL gene (aph0240) during tick transmission feeding was monitored using gene-specific primers (Kahlon et al., 2013).

Western Blotting and Confocal Microscopy

Antisera generated in this study and prior studies targeted AipA, APH0032 (Huang et al., 2010b), Asp55 (Ge et al., 2007), and Msp2 (P44) (Troese et al., 2011). Sera from an HGA patient and a dog that had been naturally infected with A. phagocytophilum were previously described (Ojogun et al., 2012). Western blot analyses (Troese et al., 2011) were performed on uninfected or A. phagocytophilum infected host cells or A. phagocytophilum DC organisms that had been subjected to surface trypsinolysis as described previously (Kahlon et al., 2013). A. phagocytophilum infected host cells were analyzed by spinning disk confocal microscopy as described (Huang et al., 2010a; Huang et al., 2012).

AipA Antiserum Inhibition of A. phagocytophilum Infection

Inhibition of host cell infection by preincubating DC organisms with heat-inactivated polyclonal antiserum targeting GST-AipA₁₋₈₇, GST-AipA₂₄₉₋₃₅₅, GST-OmpA (Ojogun, 2012), GST-Asp14 (Kahlon et al., 2013), AipA₉₋₂₁, AipA₆₁₋₈₄, AipA₁₆₅₋₁₈₂, or AipA₁₈₃₋₂₀₁ (2 mg mL⁻¹) was assessed as previously described (Ojogun et al., 2012). Serum against GST alone and preimmune serum served as negative controls. In instances where DC bacteria were incubated with combinations of antisera targeting AipA, Asp14, and/or OmpA, each respective antiserum was at a concentration of 2 mg mL⁻¹, and control antiserum was matched accordingly. Following antibody treatment, bacterial adhesion to and infection of HL-60 cells were monitored using spinning disk confocal microscopy (Ojogun et al., 2012).

Binding of GST-AipA to Mammalian Host Cells and Competitive Inhibition of A. phagocytophilum Infection

Mammalian host cells were incubated with 4 μM GST, GST-AipA₁₋₈₇, or GST-AipA₂₄₉₋₃₅₅ for 1 h at 37° C. Spinning disk confocal microscopy and flow cytometry were used to assess the binding of recombinant proteins to host cells and competitive inhibition of A. phagocytophilum infection as previously described (Ojogun et al., 2012; Kahlon et al., 2013). Assessment of the relevance of AipA to A. phagocytophilum adherence to PSGL-1 CHO cells

To determine if AipA was important for A. phagocytophilum recognition of sLe^(x)-capped PSGL-1, DC organisms were incubated with antiserum targeting AipA₁₋₈₇, AipA₂₄₉₋₃₅₅, OmpA (Ojogun et al., 2012), or preimmune control serum as described above. Next, the treated bacteria were incubated with PSGL-1 CHO cells or untransfected CHO cells for 1 h, followed by two rounds of washing with PBS to remove unbound bacteria, and enumeration of bound organisms using spinning disk confocal microscopy as described (Troese et al., 2009). As positive controls for inhibition of bacterial adherence to sLe^(x)-capped PSGL-1, PSGL-1 CHO cells were incubated with the PSGL-1 N-terminus-specific antibody, KPL-1 (BD Biosciences, San Jose, Calif.), or the sLe^(x)-specific antibody, CSLEX1 (BD Biosciences) for 30 min prior to the addition of bacteria. Mouse IgG and mouse IgM served as isotype controls for KPL-1 and CSLEX1, respectively.

Statistical Analyses

One-way analysis of variance (ANOVA) was performed using the Prism 5.0 software package (Graphpad; San Diego, Calif.) to assess statistical significance as described (Ojogun et al., 2012). Statistical significance was set at P<0.05.

Results

A. phagocytophilum Differentially Expresses AipA During the Infectious Stage of its Biphasic Developmental Cycle, the Transmission Bloodmeal of Infected Ticks, and Infection of Mammalian Hosts

AipA is a 355-amino acid (36.9 kDa) protein and a putative OMP (Nelson et al., 2008). In silico analysis of the AipA amino acid sequence predicted that residues 107 to 127, 136 to 155, and 220 to 243 form transmembrane domains that position residues 1 to 106 and 156 to 219 on the bacterial surface (FIG. 1A). Protein BLAST searches revealed that AipA does not display high sequence identity to sequenced proteins of other organisms, including Anaplasmataceae and Rickettsiales members. Because A. phagocytophilum proteins encoded by genes that are upregulated late during the biphasic developmental cycle are important for infection (Huang et al., 2010b; Troese et al., 2011; Mastronunzio et al., 2012), AipA expression throughout the infection cycle in human promyelocytic HL-60 cells was examined. aipA mRNA levels were approximately 10- to 20-fold higher between 30 and 36 hours—time points that correspond to RC-to-DC transition, exit, and reinfection—than between 4 and 26 hours, time points that correspond to conversion to and replication of non-infectious RC organisms (Troese et al., 2009) (FIG. 2A). Also, the aipA mRNA level of the DC inoculum was comparable to that detected at 36 h, a time point that correlates with reinfection.

Next, whether or not the differential aipA transcription pattern observed in infected HL-60 cells correlated with differential AipA protein expression was examined. AipA amino acids 1-87 (AipA₁₋₈₇), 165-204 (AipA₁₆₅₋₂₀₄) and 249-355 (AipA₂₄₉₋₃₅₅) contain segments that are hydrophilic and predicted to be accessible on the protein's surface (FIG. 1B). AipA₁₋₈₇ and AipA₁₆₅₋₂₀₄ are predicted to be exposed on the bacterial surface, while AipA₂₄₉₋₃₅₅ is not (FIG. 1B). AipA₁₋₈₇ and AipA₂₄₉₋₃₅₅ were expressed in Escherichia coli as proteins N-terminally fused to glutathione-S-transferase. Despite numerous attempts, soluble GST-tagged full-length AipA and AipA₁₆₅₋₂₀₄ could not be expressed (data not shown). Mouse antiserum raised against AipA₁₋₈₇ and AipA₂₄₉₋₃₅₅ fusion proteins recognized a band of the expected size in lysates of A. phagocytophilum infected but not uninfected HL-60 cells (FIG. 2B). Anti-AipA₂₄₉₋₃₅₅ recognized an additional band having an apparent mobility that was slightly smaller than 75 kDa, suggesting that AipA may dimerize. Alternatively, anti-AipA₂₄₉₋₃₅₅ may recognize an epitope that is shared with or is similar in sequence to that of the unknown A. phagocytophilum protein. An AipA₁₋₈₇ antibody was used to screen infected HL-60 cells by immunofluorescence microscopy. Consistent with the transcriptional data, approximately 70% and 96% of the A. phagocytophilum inclusions contained AipA-expressing bacteria at 28 and 32 h, respectively, whereas considerably fewer inclusions were AipA-positive at earlier time points (FIG. 2C). These data demonstrate that AipA is both transcriptionally and translationally upregulated during periods when A. phagocytophilum converts to and is in its infectious DC morphotype.

A. phagocytophilum genes that are induced during the tick transmission bloodmeal, such as ompA and asp14, encode proteins that are important for establishing infection in mammalian hosts (Ojogun et al., 2012; Kahlon et al., 2013). Therefore, aipA expression in the salivary glands of A. phagocytophilum infected I. scapularis nymphs was examined over the course of transmission feeding on naïve mice. aipA mRNA was undetectable in unfed infected ticks (FIG. 2D). However, aipA transcripts were significantly induced at 24 h of tick feeding and were increasingly expressed through to 72 h. To ensure that A. phagocytophilum transcription was not globally upregulated during tick transmission feeding, groEL (aph0240) expression was also examined. groEL was detected at the highest level in infected unfed nymphs and decreased in expression over the duration of the bloodmeal. Thus, A. phagocytophilum specifically induces select genes, including aipA, as it adapts during the transmission bloodmeal to colonize the mammalian host.

Consistent with a prior report that A. phagocytophilum preferentially expresses aipA during growth in HL-60 cells versus I. scapularis embryo-derived ISE6 cells (Nelson et al., 2008), AipA₁₋₈₇ antibody failed to detect a protein of the expected size in lysates of ISE6 cells in which A. phagocytophilum had been continually passaged for one, six, or seven weeks (FIG. 2E). APH0032, which is an A. phagocytophilum protein that was previously demonstrated to be expressed during infection of ISE6 cells (Huang et al., 2010b), was detected in all infected samples. HGA patient serum and serum from a dog that had been naturally infected with A. phagocytophilum each detected GST-AipA₁₋₈₇ (FIG. 2F), confirming that AipA is expressed and elicits a humoral immune response during A. phagocytophilum infection of humans and dogs. Two additional HGA patient sera recognized GST-AipA₁₋₈₇ (data not shown).

AipA is an A. phagocytophilum Surface Protein

To confirm whether AipA localizes to the bacterial outer membrane, confocal microscopy was used to screen infected RF/6A endothelial cells with AipA₁₋₈₇ antibody in conjunction with antiserum targeting the A. phagocytophilum major surface protein, Msp2 (P44) (Carlyon, 2012). Both antibodies detected intravacuolar organisms, yielding the ring-like staining pattern on their peripheries that is characteristic for Msp2 (P44) and other confirmed A. phagocytophilum OMPs (Ge et al., 2007; Ojogun et al., 2012; Kahlon et al., 2013) (FIG. 3A). Msp2 (P44) signal colocalized with and extended beyond the AipA signal. Next, to determine if immunoaccessible AipA domains are exposed on the bacterial surface, the surfaces of intact, host cell-free DC bacteria were treated with trypsin, an approach that has been used to confirm surface localization of A. phagocytophilum and Chlamydia trachomatis OMPs (Wang et al., 2006; Ojogun et al., 2012; Kahlon et al., 2013). AipA residues 1 to 87 include six lysine and three arginine residues, making this putative surface exposed region susceptible to tryptic digest. If AipA₁₋₈₇ or portions thereof were exposed on the A. phagocytophilum surface, then incubating intact bacteria with trypsin should result in proteolytic cleavage of this region of the protein, which, in turn, would result in an inability to detect AipA. Trypsin-treated DC organisms were solubilized, Western-blotted, and probed with antiserum specific for AipA₁₋₈₇ or a confirmed surface-exposed epitope of Asp55 (55-kDa A. phagocytophilum surface protein) (Ge et al., 2007). Blots were also probed with antiserum targeting APH0032, which does not localize to the A. phagocytophilum outer membrane (Huang et al., 2010b). After surface trypsinolysis, APH0032 was detected but AipA and Asp55 were not (FIG. 3B). Thus, AipA residues 1 to 87 are exposed on the A. phagocytophilum surface.

GST-AipA Requires Amino Acids 1 to 87 to Bind and Competitively Inhibit A. phagocytophilum Infection of Mammalian Host Cells

Since AipA is an exposed surface protein that is induced during key infection stages in the A. phagocytophilum life cycle, its ability to facilitate interactions with mammalian host cell surfaces to promote infection was investigated. It was assessed if GST-tagged AipA₁₋₈₇ and AipA₂₄₉₋₃₅₅ bind to RF/6A cells. GST alone served as a negative control. GST antibody detected GST-AipA₁₋₈₇ that had adhered to the host cells by both immunofluorescence microscopy and flow cytometry (FIG. 4, A and B). GST-AipA₂₄₉₋₃₅₅ bound poorly, at best, and GST alone did not bind to host cells. Based on their differential adhesion capabilities, it was rationalized that GST-AipA₁₋₈₇ but not GST-AipA₂₄₉₋₃₅₅ would be able to serve as a competitive agonist to inhibit A. phagocytophilum infection. Indeed, preincubating HL-60 and RF/6A cells with GST-AipA₁₋₈₇ significantly reduced the percentages of infected cells and the number of bacterial inclusions per cell by nearly four-fold relative to incubation with GST alone (FIG. 4, C to F). In contrast, GST-AipA₂₄₉₋₃₅₅ did not inhibit A. phagocytophilum infection of HL-60 cells and reduced infection of RF/6A cells by only a small degree. These data suggest that AipA residues 1 to 87 contain a domain that contributes to A. phagocytophilum invasion of myeloid and endothelial cells.

Antiserum Targeting AipA Residues 1 to 87 Inhibits A. phagocytophilum Infection of Host Cells

Given that AipA amino acids 1 to 87 are exposed on the A. phagocytophilum surface and contribute to infection, it was assessed if treating DC organisms with heat-inactivated AipA₁₋₈₇ antiserum prior to incubating them with HL-60 cells would inhibit infection. OmpA antiserum, for which its ability to inhibit A. phagocytophilum infection was previously validated (Ojogun et al., 2012), was a positive control. Anti-AipA₁₋₈₇ and anti-OmpA each reduced the number of infected cells and the number of bacterial inclusions per cell by approximately 40% (FIG. 5, A and B). In contrast, AipA₂₄₉₋₃₅₅ antiserum had no effect on A. phagocytophilum infection. Consistent with the studies of A. phagocytophilum invasins (Ojogun et al., 2012; Kahlon et al., 2013), neither AipA₁₋₈₇ nor OmpA antiserum inhibited bacterial adhesion to HL-60 cells (data not shown).

AipA Targets a sLe^(x)-Capped PSGL-1-Independent Receptor

sLe^(x)-capped PSGL-1 is the only known A. phagocytophilum receptor on myeloid host cells (Herron et al., 2000), and OmpA binds the α2,3-sialic acid determinant of sLe^(x) (Ojogun et al., 2012). Since A. phagocytophilum interactions with sLe^(x)-capped PSGL-1 involve at least one bacterial surface protein in addition to OmpA (Carlyon et al., 2003; Yago et al., 2003; Reneer et al., 2006; Sarkar et al., 2007; Reneer et al., 2008; Ojogun et al., 2012), it was investigated if AipA₁₋₈₇ antiserum could inhibit bacterial binding to Chinese hamster ovary (CHO) cells transfected to express sLe^(x)-capped PSGL-1 (PSGL-1 CHO cells). These cells are excellent models for studying A. phagocytophilum interactions with sLe^(x)-capped PSGL-1 as they, but not untransfected CHO cells that do not express the receptor, support bacterial binding (Carlyon et al., 2003; Xia et al., 2003; Yago et al., 2003; Reneer et al., 2006; Sarkar et al., 2007; Reneer et al., 2008; Troese et al., 2009). DC organisms were incubated with AipA₁₋₈₇ or AipA₂₄₉₋₃₅₅ antiserum prior to being added to PSGL-1 CHO cells. Bacteria pretreated with preimmune serum were a negative control, whereas bacteria pretreated with OmpA antiserum served as a positive control. Additional positive controls for blocking A. phagocytophilum adhesion were PSGL-1 CHO cells that had been pretreated with KPL-1 or CSLEX1, which are monoclonal antibodies that block the bacterium's access to the PSGL-1 N-terminus or the α2,3-linked sialic acid determinant of sLe^(x), respectively (Goodman et al., 1999; Herron et al., 2000; Troese et al., 2009). Incubating DC organisms with OmpA antibody and incubating PSGL-1 CHO cells with KPL-1 or CSLEX1 significantly reduced the numbers of bound DC organisms by two- to three-fold (FIG. 5C). A. phagocytophilum bound poorly to untransfected CHO cells. Preimmune serum, anti-AipA₁₋₈₇, and anti-AipA₂₄₉₋₃₅₅ failed to inhibit bacterial binding to PSGL-1 CHO cells. Therefore, AipA contributes to A. phagocytophilum cellular invasion by interacting with an sLe^(x)-capped PSGL-1-independent receptor.

A Combination of Antisera Targeting AipA, OmpA, and Asp14 Blocks A. phagocytophilum Infection of Host Cells

A. phagocytophilum infection requires cooperative interactions of multiple invasins with the host cell surface (Truchan et al., 2013). Incubating DC organisms with antiserum targeting full-length OmpA (Ojogun et al., 2012), full-length Asp14 (Kahlon et al., 2013), or AipA₁₋₈₇ significantly, but only partially, reduced A. phagocytophilum infection of mammalian host cells. It was therefore investigated if blocking multiple bacterial-host interactions by treating DC organisms with combinations of AipA₁₋₈₇, OmpA, and/or Asp14 antisera could improve blocking efficacy. The result was synergistic: whereas antisera combinations targeting two of the three invasins together more effectively inhibited infection than an antiserum targeting an individual protein, the most effective blocking of A. phagocytophilum infection was achieved using antisera against all three invasins (FIG. 6). These data demonstrate that simultaneously targeting AipA, OmpA, and Asp14 is an effective means for preventing or treating A. phagocytophilum infection.

The AipA Invasion Domain is Contained within Residues 9 to 21

It was next sought to pinpoint the AipA invasion domain. The competitive agonist and antisera blocking studies indicated that this domain lies within residues 1 to 87. Based on hydrophobicity and surface probability analyses (FIG. 1B), it was rationalized that, of the AipA region of interest, amino acids 9 to 21 and/or 61 to 84 were most likely to facilitate interactions with host cells that promote infection. Accordingly, rabbit antisera against each of these peptides was generated for use in antibody blocking experiments. The AipA region encompassed by residues 165 to 204 (AipA₁₆₅₋₂₀₄) is predicted to be exposed on the A. phagocytophilum outer membrane, hydrophilic, and accessible on the surface of AipA (FIG. 1B). Yet, the contribution of AipA₁₆₅₋₂₀₄ to infection was unknown due to our inability to express it as a soluble recombinant protein. Therefore, antisera to peptides corresponding to AipA amino acids 165 to 182 and 183 to 201 was also raised. Each AipA peptide antiserum recognized endogenous AipA in lysates of A. phagocytophilum infected, but not uninfected HL-60 cells and was specific for the peptide against which it was raised (FIG. 7, A and B). Incubating DC organisms with AipA₉₋₂₁ antiserum reduced infection of HL-60 cells by approximately 47% relative to preimmune control serum, an inhibitory effect that was analogous to the reduction achieved using antiserum against AipA₁₋₈₇ or OmpA (FIG. 7C). Antisera against each of the other three AipA peptides and AipA₂₄₉₋₃₅₅ had minimal or no inhibitory effect on infection. Thus, the AipA invasion domain is contained within residues 9 to 21.

Discussion

Invasins of obligate intracellular pathogens are dualistic: they are essential for bacterial entry into host cells but, as such, they are also “Achilles' Heels” that can be blocked to prevent infection and pathogen survival. Targeting invasins of arthropod-transmitted pathogens that are induced during the arthropod blood meal is an attractive approach because of its ability to prevent not only establishment of infection but also disease transmission. Given that A. phagocytophilum infection is predicated on the cooperative actions of multiple bacterial surface-associated invasins (Truchan et al., 2013), most of which are induced during the tick transmission bloodmeal (Ojogun et al., 2012; Kahlon et al., 2013), effective prophylaxis against granulocytic anaplasmosis can be achieved by identifying and targeting these invasins. AipA is an attractive target to include in a multicomponent granulocytic anaplasmosis vaccine. It is an invasin that is induced during tick transmission feeding, is preferentially expressed during the bacterium's infectious stage, and functions synergistically with OmpA and Asp14 to mediate optimal infection of mammalian host cells.

The exposure of AipA on the infectious DC form surface makes it accessible to blocking antibodies. Indeed, preincubating DC organisms with AipA antiserum significantly reduced infection of HL-60 cells. Pretreatment of DC bacteria with a combination of antibodies targeting AipA, OmpA, and Asp14 nearly abolished infection, whereas pretreatment with antibodies against one or two of the three invasins was less effective. Thus, AipA, OmpA, and Asp14 are collectively critical for infection and targeting all three together blocks infection in vitro. Spotted fever group Rickettsia species, which are in the Order Rickettsiales with A. phagocytophilum, also use multiple surface proteins to promote entry into host cells (Martinez et al., 2004; Cardwell et al., 2009; Chan et al., 2009; Chan et al., 2010; Riley et al., 2010). Moreover, this pathogenic strategy is common among numerous other Gram negative bacterial pathogens, including Chlamydia pneumoniae (Molleken et al., 2010; Molleken et al., 2013), Legionella pneumophila (Garduno et al., 1998; Stone et al., 1998; Cirillo et al., 2001; Chang et al., 2005; Vandersmissen et al., 2010; Duncan et al., 2011), Bordetella pertussis (Brennan et al., 1996), Haemophilus influenzae (Jurcisek et al., 2007; Giufre et al., 2008; Chang et al., 2011; Dicko et al., 2011; Jalalvand et al., 2013; Singh et al., 2013) and Leptospira species (Barbosa et al., 2006; Pinne et al., 2010; Verma et al., 2010; Zhang et al., 2012).

GST-AipA is capable of binding to mammalian cells, which suggests that it functions not only as an invasin but also as an adhesin. Yet, AipA antibodies and GST-AipA₁₋₈₇ each failed to inhibit A. phagocytophilum binding to mammalian cells. In these experiments, the role of AipA as an adhesin was presumably masked by the presence of other adhesins/invasins, such as OmpA and Asp14, on the bacterial surface (Ojogun et al., 2012; Kahlon et al., 2013). The AipA receptor is unknown. However, because AipA antibody failed to inhibit A. phagocytophilum binding to PSGL-1 CHO cells, it can be concluded that AipA recognizes a sLe^(x)-capped PSGL-1 independent receptor. AipA and Asp14, which also engages a sLe^(x)-capped PSGL-1 independent receptor (Kahlon et al., 2013), complement the sLe^(x)-targeting activity of OmpA (Ojogun et al., 2012).

Bacterial genes that are upregulated during transmission feeding of arthropod vectors are critical for various vector-transmitted bacteria to establish infection in their mammalian hosts (Hinnebusch et al., 1996; Perry et al., 1997; Grimm et al., 2004; Tilly et al., 2006). Consistent with these phenomena, aipA is not expressed by A. phagocytophilum during its residence in ISE6 cells or I. scapularis nymphs, but is induced when the bacterium is cultivated in mammalian tissue culture cells and during tick transmission feeding. Furthermore, A. phagocytophilum expresses AipA during infection of humans and dogs. Thus, AipA is dispensable for bacterial colonization of the tick vector, but is important for infecting mammalian hosts. Similar expression profiles have been observed for the other identified invasins OmpA, Asp14, and APH1235 (Mastronunzio et al., 2012; Ojogun et al., 2012; Kahlon et al., 2013). Also, like APH1235 (Troese et al., 2011; Mastronunzio et al., 2012), AipA is pronouncedly upregulated when the bacterium is in the DC stage. In agreement with the invasive role of the DC morphotype, both proteins are important for establishing and/or maintaining infection in mammalian host cells.

How AipA is transported to and associates with the bacterial outer membrane is unclear, as it lacks a canonical signal peptide that would target it for Sec-dependent or twin-arginine secretion. This conundrum is further complicated as AipA is unique to A. phagocytophilum and bears no homology to any known crystal structure. Perhaps AipA is an atypical transmembrane protein or a peripheral membrane protein that is anchored to the bacterial outer membrane via a posttranslational modification. AipA colocalizes with the confirmed outer membrane protein, Msp2 (P44) and functions in concert with OmpA and Asp14, both of which have also been shown to colocalize with Msp2 (P44) (Kahlon et al., 2013; Ojogun et al., 2012). Msp2 (P44) has been proposed to form heteromeric complexes that mediate interactions with host cells (Park et al., 2003). Given that AipA, Asp14, and OmpA synergistically promote A. phagocytophilum infection of host cells, it will be important to determine if they do so as a multimeric invasin complex that includes Msp2 (P44).

The AipA invasion domain lies within residues 9 to 21, which is a hydrophilic region of the protein that is exposed on the bacterial surface. Antiserum targeting this span reduces A. phagocytophilum infection of host cells by a level comparable to that achieved by antiserum targeting the entire surface-exposed N-terminal domain of AipA. The observed inhibition is specific to anti-AipA₉₋₂₁, as antisera targeting peptides corresponding to all other predicted hydrophilic regions of AipA exhibited no inhibitory effect. Previously, it was discovered that the N-terminal ectodomain of OmpA is required for recognition of α2,3-linked sialic acid of sLe^(x) (Ojogun et al., 2012) and the Asp14 invasion domain lies within residues 101-124 (Kahlon et al., 2013).

Granulocytic anaplasmosis can be debilitating or fatal, and there is no vaccine that protects against the disease. The results described herein show for the first time that simultaneously targeting multiple A. phagocytophilum invasins effectively blocks infection in vitro.

Example 2. Essential Domains of Anaplasma phagocytophilum Invasins Utilized to Infect Mammalian Host Cells

Summary

Anaplasma phagocytophilum causes granulocytic anaplasmosis, an emerging disease of humans and domestic animals. The obligate intracellular bacterium uses its invasins OmpA, Asp14, and AipA to infect myeloid and non-phagocytic cells. The present study identifies the OmpA binding domain as residues 59 to 74. Polyclonal antibody generated against a peptide spanning OmpA residues 59 to 74 inhibited A. phagocytophilum infection of host cells and binding to its receptor, sialyl Lewis x (sLe^(x))-capped P-selectin glycoprotein ligand 1. Molecular docking analyses predicted that OmpA residues G61 and K64 interact with the two sLe^(x) sugars that are important for infection, α2,3-sialic acid and α1,3-fucose. Amino acid substitution analyses demonstrated that K64 was necessary and G61 was contributory for recombinant OmpA to bind to host cells and competitively inhibit A. phagocytophilum infection. Adherence of OmpA to RF/6A endothelial cells, which express little to no sLe^(x) but express the structurally similar glycan, 6-sulfo-sLe^(x), required α2,3-sialic acid and α1,3-fucose and was antagonized by 6-sulfo-sLe^(x) antibody. Binding and uptake of OmpA-coated latex beads by myeloid cells was sensitive to sialidase, fucosidase, and sLe^(x) antibody. The Asp14 binding domain was also defined, as antibody specific for residues 113 to 124 inhibited infection. An antibody cocktail targeting the OmpA, Asp14, and AipA binding domains neutralized A. phagocytophilum binding and infection of host cells. This study dissects OmpA-receptor interactions and demonstrates the effectiveness of binding domain-specific antibodies for blocking A. phagocytophilum infection.

Introduction

Human granulocytic anaplasmosis (HGA) is an emerging tick-borne zoonosis in the United States, Europe, and Asia (Truchan et al., 2013). The number of HGA cases reported to the U. S. Centers for Disease Control and Prevention rose nearly seven-fold between 2003 and 2012 (CDC 2013; Hopkins et al., 2005). Seroprevalence data indicate that the disease is underreported in some endemic regions (Hao et al., 2013; Zhang et al., 2012; Zhang et al., 2009; Aguero et al., 2002; Bakken et al., 1998). HGA can also be spread via perinatal, nosocomial, and blood transfusion routes (Bakken et al., 1998; Alhumaidan et al., 2013; Annen et al., 2012; Jereb et al., 2012). It is an acute illness characterized by fever, chills, headache, malaise, leukopenia, thrombocytopenia, and elevated liver enzymes. Complications can include shock, seizures, pneumonitis, rhabdomyolysis, hemorrhage, increased susceptibility to secondary infections, and death. Risk for complications and fatality is greater for the elderly, the immunocompromised, and when proper diagnosis and/or antibiotic therapy are delayed (Truchan et al., 2013). The causative agent of HGA is Anaplasma phagocytophilum, an obligate intracellular bacterium that exhibits a tropism for neutrophils (Truchan et al., 2013). A. phagocytophilum is carried by a variety of wild animal reservoirs and, in addition to humans, causes disease in domestic animals including dogs, cats, horses, and sheep (Stuen et al., 2013).

A. phagocytophilum exhibits a biphasic developmental cycle similar to that of Chlamydia spp., Ehrlichia spp., and Coxiella burnetii (Bastidas et al., 2013; Minnick et al., 2012; Zhang et al., 2007; Troese et al., 2009). The A. phagocytophilum infectious dense-cored (DC) form promotes its receptor-mediated uptake into a host cell derived vacuole. Within its vacuole, the DC develops into the non-infectious reticulate cell (RC) form that replicates to form a bacterial cluster called a morula (Troese et al., 2009; Ojogun et al., 2012). RCs then convert back to DCs and are released to initiate the next infection cycle (Troese et al., 2009).

Sialyl Lewis x ([NeuAcα(2-3)Galβ1-4(Fucα1-3)GlcNac]; sLe^(x)), an α2,3-5 sialylated and α1,3-fucosylated core-2 O-linked glycan that caps the N-termini of selectin ligands (Sperandio et al., 2006), is a critical A. phagocytophilum receptor (Goodman et al., 1999). sLe^(x) is richly expressed on mammalian cells that are permissive for A. phagocytophilum infection such as neutrophils, bone marrow progenitors, and promyelocytic HL-60 cells (Karakantza et al., 1994; Symington et al., 1985; Fukuda et al., 1984). A. phagocytophilum recognizes sLe^(x) that caps the N-terminus of P-selectin glycoprotein ligand-1 (PSGL-1) on these myeloid cells (Goodman et al., 1999; Herron et al., 2000). Neutrophils and HL-60 cells that have been treated with an sLe^(x) blocking antibody, from which surface sialic acids have been enzymatically removed, or that are devoid of sialyltransferase and/or α1,3-fucosyltransferase activity are resistant to A. phagocytophilum binding and infection (Ojugun et al., 2012; Goodman et al., 1999; Carylon et al., 2003; Yago et al., 2003). A. phagocytophilum also infects rhesus monkey choroidal (RF/6A) endothelial cells, megakaryoblastic MEG-01 cells, and bone marrow-derived mast cells in tissue culture. Infection of these non-myeloid host cell types depends on sLe^(x) itself, α2,3-sialic acid, and/or α1,3-fucose (Huang et al., 2012; Kahlon et al., 2013; Mastronunzio et al., 2012; Ojogun et al., 2011). Thus, sLe^(x) and possibly other closely related α2,3-sialylated and α1,3-fucosylated molecules are essential for efficient A. phagocytophilum infection of mammalian cells.

A. phagocytophilum OmpA and α2,3-sialic acid (N-acetylneuraminic acid [Neu5Ac], further referred to as sialic acid throughout) was identified as the bacterium's first adhesin/invasin-receptor pair (Ojogun et al., 2012). OmpA binding to the α2,3-sialic acid determinant of sLe^(x) on myeloid cells and to α2,3-sialylated glycans on RF/6A cells are vital steps in A. phagocytophilum invasion of these host cell types (Ojogun et al., 2012). Exposure of OmpA on the A. phagocytophilum DC surface makes it accessible to antibodies (Ojogun et al., 2012), which could be used to exploit the bacterium's obligatory intracellular nature to block the host cell invasion step that is essential for survival. The OmpA binding domain that recognizes α2,3-sialic acid lies within amino acids 19 to 74 (Ojogun et al., 2012), but has yet to be specifically identified. The A. phagocytophilum OMP that recognizes α1,3-fucose is unknown. As shown in Example 1, OmpA functions in concert with two additional invasins that are also upregulated during tick transmission feeding, Asp14 (14-kDa A. phagocytophilum surface protein) and AipA (A. phagocytophilum invasion protein A), to promote optimal A. phagocytophilum entry into mammalian host cells (Kahlon et al., 2013; Seidman et al., 2014). The AipA binding domain was defined in Example 1 as residues 9 to 21.

In this study, antibody blocking, in silico docking models, and site directed mutagenesis was used to identify the A. phagocytophilum OmpA binding domain, specifically the key residues that are essential for its adhesin/invasin activity, and determined that it recognizes both α2,3-sialic acid and α1,3-fucose. This work represents the most detailed study of any rickettsial adhesin/invasin-receptor pair to date. Furthermore, the Asp14 binding domain was identified and it was confirmed that an antibody cocktail targeting the binding domains of OmpA, Asp14, and AipA nearly abolishes A. phagocytophilum infection of host cells.

Materials and Methods

Cell Lines and Cultivation of A. phagocytophilum

Uninfected and A. phagocytophilum infected (NCH-1 strain) HL-60 cells (ATCC CCL-240) and RF/6A cells (ATCC CRL-1790, Manassas, Va.) were maintained as previously described (Troese et al., 2009; Huang et al., 2012). CHO (−) and PSGL-1 CHO cells were cultivated as described (Li et al., 1996).

Site Directed Mutagenesis and Recombinant Proteins

pGST-OmpA, which encodes OmpA19-205 N-terminally fused to GST, was previously constructed (Ojogun et al., 2012). Using pGST-OmpA as the template, the QuikChange® Lightning (Agilent Technologies, Santa Clara, Calif.) protocol was used per the manufacturer's guidelines to perform site-directed insertions and point mutagenesis of the ompA insert sequence. For site directed insertions, a five-amino acid insert sequence (CLNHL; SEQ ID NO:75) was selected based on previous studies that had successfully employed the linker-scanning method (Anton et al., 2004; Okoye et al., 2006), which is used to insert peptide “linkers” to disrupt protein binding domains without perturbing overall protein structure. The sequence chosen for the insertion peptide, CLNHL (SEQ ID NO:75), was a consensus sequence based on the most common amino acids at their respective positions in the insertion peptides used in prior studies (Anton et al., 2004; Okoye et al., 2006). The nucleotide sequence, 5′-TGCCTGAACCACCTG-3′ (SEQ ID NO:76), which encoded CLNHL (SEQ ID NO:75), was inserted in the ompA sequence of pGST-OmpA between ompA nucleotides 102 and 103, 162 and 163, 186 and 187, 201 and 202, 216 and 217, and 231 and 232 to yield plasmids that encoded GST-OmpA proteins bearing CLNHL inserts between OmpA amino acids 34 and 35, 54 and 55, 62 and 63, 67 and 68, 72 and 73, and 77 and 78, respectively. Likewise, the QuikChange protocol was used to perform site directed mutagenesis to yield plasmids that encoded GST-OmpA proteins having R32, D53, K60, G61, K64, K65, E69, and/or E72 converted to alanine. GST-OmpA mutants were expressed and purified as previously described (Ojogun et al., 2012). Plasmids encoding His-tagged wild type and site-directed mutant OmpA proteins were generated by amplifying wild type and mutant ompA sequences using primers containing ligase-independent cloning (LIC) tails and annealing the amplicons into the pET46 Ek/LIC vector (Novagen, EMD Millipore, Darmstadt, Del.) per the manufacturer's instructions. His-OmpA proteins were expressed and purified by immobilized metal-affinity chromatography as previously described (Miller et al., 2011).

Molecular Modeling of the OmpA-sLe^(x) Interaction

To obtain a putative three-dimensional OmpA protein structure, the mature OmpA sequence was threaded onto the solved crystal structures of proteins with similar sequences using the PHYRE2 server as previously described (Ojogun et al., 2012). Amino acids 19 to 150 (73% of the mature OmpA sequence) were modeled with greater than 90% confidence to known structures for similar proteins (Protein Data Bank [PDB] files 2aiz [Haemophilus influenzae OmpP6 peptidoglycan associated lipoprotein (PAL)], 4g4v [Acinetobacter baumanni PAL], 4b5c [Burkholderia pseudomallei PAL], 3ldt [Legionella pneumophila OmpA], 2kgw [Mycobaterium tuberculosis OmpATb]). The remainder of the protein lacked sufficient homology to any experimentally derived structure, but could be modeled using the Poing method (Kelley et al., 2009), which was performed as part of the Phyre2 analyses. The sLe^(x)-PSGL-1peptide (residues 61 to 77) and the sLe^(x) glycan itself was extracted from the solved crystal structure of PSGL-1 (PDB 1G1S) in PyMol and saved as an individual PDB file. Open Babel software was used to convert PDB files to PDBQT (Protein Data Bank, Partial Charge and Atom Type) format in order to perform OmpAsLe^(x) docking analysis. AutoDock Tools software was used to generate the docking output files for both the OmpA protein structure and the sLe^(x) ligand. The search location for OmpA was generated in AutoDock Tools by setting a search grid that encompassed OmpA residues 19 to 74. Molecular docking was performed using AutoDock Vina to identify potential points of interaction between OmpA and sLe^(x). The top two OmpA-sLe^(x) models generated by AutoDock Vina had the same predicted affinity value of −4.2 kcal/mol and were selected for analysis in PyMol to determine potential points of contact.

Antibodies, Reagents, Enzyme-Linked Immunosorbent Assay (ELISA), and Western Blotting

To generate antisera specific to the OmpA and Asp14 binding domains, peptides corresponding to OmpA residues 23 to 40, 41 to 58, and 59 to 74 and Asp14 residues 98 to 112 and 113 to 124 were synthesized, conjugated to keyhole limpet hemocyanin, administered to rabbits, and the resulting OmpA and Asp14 peptide-specific sera were affinity-purified by New England Peptide (Gardner, Mass.). Each peptide antiserum's specificity for the peptide against which it had been raised and for its protein target was determined by ELISA using the TMB substrate kit (Thermo Scientific, Waltham, Mass.) following the manufacturer's instructions or by Western blot analysis as previously described (Carylon et al., 2002). Mouse anti-AipA peptide antisera have been previously described (Seidman et al., 2014). sLe^(x) 564 antibodies CSLEX1 (BD Biosciences, San Jose, Calif.) and KM93 (Millipore, Darmstadt, Del.) and PSGL-1 N-terminus-specific antibody 565 KPL-1 (BD Biosciences) were obtained commercially. Fab fragments of OmpA and Asp14 peptide-specific antisera were generated using the Fab Preparation Kit (Pierce, Rockford, Ill.) according to the manufacturer's instructions. His tag and Alexa Fluor 488-conjugated secondary antibodies and Alexa Fluor 488-conjugated streptavidin were obtained from Invitrogen (Carlsbad, Calif.). Biotinylated AAL and MAL II were obtained from Vector Labs (Burlingame, Calif.). Glycosidases used in this study were α2,3/6-sialidase (Sigma-Aldrich, St. Louis, Mo.) and α1,3/4-fucosidase (Clontech, Mountain View, Calif.).

Sequence Alignments

The NCH-1 gene sequence for ompA (APH0338) was previously determined (Ojogun et al., 2012; Kahlon et al., 2013; Seidman et al., 2014). A Protein BLAST (basic local alignment search tool) search using the NCH-1 OmpA predicted protein sequence as the query was used to identify homologs in other Anaplasmataceae species and in A. phagocytophilum strains HZ (Rikihisa et al., 1997), HGE1 (Goodman et al., 1996), Dog (Al-Khedery et al., 2012), JM (Johnson et al., 2011), MRK (Madigan et al., 1987; Gribble, 1969), CRT35, CRT38 (Massung et al., 2007), and NorV2 (Al-Khedery et al., 2012), for which the genomes are available. All of these strains except for NorV2 had been originally isolated from clinically affected humans and animals. HZ and HGE1 were recovered from human patients in Westchester, N.Y., USA and Minnesota, USA, respectively. The Dog and JM strains were isolated from a dog in Minnesota, USA and a meadow jumping mouse (Zapus hudsonius) in Camp Ripley, Minn., USA. MRK had been recovered from a horse in California, USA. CRT35 and CRT38 are isolates of the A. phagocytophilum Ap-variant 1 strain that were recovered from ticks collected at Camp Ripley, Minn., USA. NorV2 is a naturally occurring A. phagocytophilum isolate that was maintained in an experimentally infected lamb, exhibits reduced virulence in sheep, and differs in its 16S rRNA gene sequence when compared to other sheep isolates. OmpA sequence alignments were generated using Clustal W.

Binding of Recombinant OmpA Proteins to Host Cells

For binding of His- or GST-tagged OmpA proteins to host cells, RF/6A or HL-60 cells were incubated with 4 μM recombinant protein in culture media for 1 h in a 37° C. incubator supplemented with 5% CO2 and a humidified atmosphere. To assess for the presence of sLe^(x) or 6-sulfo-sLe^(x) on RF/6A cell surfaces, the cells were fixed in 4% PFA in PBS for 1 h at room temperature followed by incubation with CSLEX1, KM93, or G72 for 1 h at room temperature. Antibody incubations and washes were performed as described previously (Reneer et al., 2006). Spinning-disk confocal microscopy using an Olympus BX51 microscope affixed with a disk-spinning unit (Olympus, Center Valley, Pa.) and/or flow cytometry using a BD FACS Canto™ II (BD Biosciences) were performed to assess binding of antibodies or His-OmpA proteins to host cell surfaces as previously described (Ojogun et al., 2012; Kahlon et al., 2013). In some cases, RF/6A cells were pretreated with α2,3/6-sialidase, α1,3/4-fucosidase, AAL, MAL II, or sLe^(x)- or 6-sulfo-sLe^(x)-specific antibodies prior to incubation with His-OmpA.

Competitive Inhibition of A. phagocytophilum Binding and Infection

Competitive inhibition assays utilizing recombinant protein or antibody were performed and analyzed by spinning-disk confocal microscopy as previously described (Ojogun et al., 2012; Kahlon et al., 2013). To determine if A. phagocytophilum binding to PSGL-1 CHO cells or infection of RF/6A cells involved bacterial binding to host cell surface fucose residues, the host cells were treated with α1,3/4-fucosidase (10 μU/mL) prior to the addition of DC organisms and assessment for bacterial binding or infection as previously described (Troese et al., 2009; Ojogun et al., 2012). For competitive inhibition assays using antisera raised against OmpA or Asp14 peptides, A. phagocytophilum DC bacteria were incubated with serially diluted concentrations of antiserum. Preimmune rabbit serum (200 μg/mL) was a negative control. Assays using combinations of two or three different OmpA, Asp14, or AipA peptide antibodies were performed using 100 μg/mL per antibody. Preimmune serum (200 μg/mL or 300 μg/mL, based on the combined total of peptide antisera) served as a negative control. Competitive inhibition assays using OmpA and/or Asp14 Fab fragments were performed exactly as described for antisera. Preimmune Fab fragments served as a negative control.

OmpA Coated Bead Uptake Assay

1.8×107 red fluorescent sulfate-modified 1.0 μm diameter microfluorospheres (Life Technologies, Carlsbad, Calif.) were mixed by rotation with 8 μg of His-OmpA, or His-OmpA proteins bearing alanine substitutions, in 400 μL of 50 mM phosphate buffered saline (PBS) supplemented with 0.9% NaCl at room temperature overnight in the absence of light. The His-OmpA coated beads were centrifuged at 5,000 g for 25 min, followed by three washes in 50 mM PBS. Coated beads were resuspended in 400 μL of 50 mM PBS, 0.9% NaCl, 1% BSA and stored at 4° C. until use. To validate that the beads were conjugated with His-OmpA, 1.8×104 of the beads were screened by immunofluorescent microscopy using mouse polyclonal OmpA antisera followed by Alexa Fluor 488-conjugated goat anti-mouse IgG as described (Ojogun et al., 2012). To assess binding to and uptake by HL-60 or RF/6A cells, His-OmpA coated beads or uncoated control beads were resuspended in the appropriate culture medium and added to host cells at a concentration of 500 beads/cell. For adherent RF/6A cells, beads were centrifuged onto the host cells at 1,000 g for 5 min. The cells plus beads were incubated for 1 h at 37° C. in a 5% CO₂ supplemented humidified incubator followed by washing the cells three times with PBS to remove unbound beads. Non-adherent HL-60 cells were mixed with the beads in suspension, incubated as described above, and three PBS washes were performed intermittently between five-min spins performed at 300 g. To assess binding, the host cells were fixed in 4% paraformaldehyde (PFA) in PBS, mounted with ProLong® Antifade Gold gel mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen), and analyzed by spinning-disk confocal microscopy as previously described (Ojogun et al., 2012). For uptake assays, after the final wash, the host cells were resuspended in culture medium and cultivated for an additional 7 h. The cells were washed three times in PBS, incubated with a 0.25% trypsin solution (Hyclone™, Thermo Scientific, Waltham, Mass.) for 10 min at 37° C. to cleave host cell surface proteins and consequently remove noninternalized beads, and washed three times with PBS. HL-60 cells were cytospun onto glass microscope slides and fixed, mounted, and screened as described above. RF/6A cells were added to wells containing coverslips, incubated overnight in a 37° C. incubator supplemented with 5% CO₂ and a humidified atmosphere to allow the host cells to adhere prior to further processing. To determine if His-OmpA coated bead binding or uptake was temperature sensitive, some experiments were performed at 4° C. To assess the contribution of sLe^(x) or PSGL-1 determinants to His-OmpA coated bead binding and uptake, host cells were pretreated with α2,3-sialidase (5 μg/mL), α1,3/4-fucosidase (10 μU/mL), sLe^(x)-specific antibody CSLEX1 (10 μg/mL), PSGL-1 N-terminus-specific antibody KPL-1 (10 μg/mL), or vehicle or isotype controls as previously described (Ojogun et al., 2012) prior to the bead binding and uptake assays.

Scanning Electron Microscopy

Coverslips of RF/6A cells were incubated with OmpA coated or control beads as described above. The coverslips were fixed in 2.0% glutaraldehyde in 0.1 M sodium cacodylate for 1 h at room temperature. The coverslips were subjected to two 10-min washes in 0.1 M sodium cadodylate and fixed in 1.0% osmium tetroxide in 0.1 M sodium cacodylate for 1 h. The coverslips were rinsed two more times with 0.1 M sodium cadodylate buffer for 10 min each. The samples were dehydrated by successive 5-min incubations in 50% ethanol, 70% ethanol, 80% ethanol, 95% ethanol, and three 10-min washes in 10% ethanol. Next, the samples were incubated three times for 30 min each in hexamethyldisilazane, air-dried, mounted with silver paint, and sputter coated with gold before imaging on a Zeiss EVO® 50XVP scanning electron microscope (Thornwood, N.Y.). For HL-60 cells incubated in suspension with beads, the samples were retained on a 0.1 μm filter and processed exactly as described for RF/6A cells.

Statistical Analyses

The Prism 5.0 software package 680 (Graphpad, San Diego, Calif.) was used to determine the statistical significance of data using one-way analysis of variance (ANOVA) or the Student's T-test, as previously described (Ojogun et al., 2012). Statistical significance was set to P<0.05.

Results

OmpA Amino Acids 59 to 74 are Critical for A. phagocytophilum to Bind to sLe^(x) Capped PSGL-1 and for Infection of Mammalian Host Cells

The OmpA region that is important for A. phagocytophilum infection of mammalian host cells lies within residues 19 to 74 (OmpA19-74) (Ojogun et al., 2012). As a first step in further delineating the binding domain, polyclonal antisera against peptides corresponding to OmpA amino acids 23 to 40, 41 to 58, and 59 to 74 were raised. It was verified that the antisera were specific for OmpA by confirming that each recognized recombinant forms of mature OmpA (minus the signal sequence; corresponding to residues 19 to 205 and hereafter referred to as OmpA) and OmpA19-74, but neither OmpA75-205 nor Asp14 (FIG. 8A). Anti-OmpA41-58 and anti-OmpA59-74 were specific for their target peptides at all serum dilutions. Anti-OmpA23-40 was specific for its target peptide at most dilutions tested, but exhibited low level recognition of OmpA41-58 at dilutions below 1:12,800 (FIG. 8A). Next, it was evaluated if any of the OmpA peptide antisera could inhibit A. phagocytophilum infection of host cells. Bacteria that had been treated with anti-OmpA or preimmune serum served as positive and negative controls, respectively. As previously observed (Ojogun et al., 2012), OmpA antibody reduced the percentage of A. phagocytophilum infected HL-60 cells by approximately 40% (FIG. 8B). OmpA59-74 antibody exhibited a dose-dependent inhibitory effect and, at a concentration of 200 ug/ml, reduced the percentage of infected HL-60 cells by approximately three-fold. Antisera targeting OmpA residues 23 to 40 and 41 to 58 exhibited very little to no inhibition of infection, regardless of concentration. Unless otherwise specified, all antisera were used at a concentration of 200 ug/ml in subsequent blocking experiments.

sLe^(x)-capped PSGL-1 is an A. phagocytophilum receptor on human myeloid cells (Goodman et al., 1999), and OmpA has been shown to bind the sLe^(x) portion (Ojogun et al., 2012). Because OmpA59-74antibody significantly inhibited A. phagocytophilum infection of HL-60 cells, it was rationalized that OmpA amino acids that are critical for engaging the receptor are within residues 59 to 74. To test this hypothesis, the abilities of antisera targeting various portions of OmpA to interfere with A. phagocytophilum binding to Chinese hamster ovary cells transfected to express sLe^(x)-capped PSGL-1 (PSGL-1 CHO cells) was assessed. These cells are useful models for studying A. phagocytophilum interactions with sLe^(x) and/or PSGL-1 because they robustly support bacterial binding but not infection, while untransfected CHO [CHO (−)] cells that lack expression of these receptors poorly support bacterial binding. Anti-OmpA59-74 reduced the mean number of bound A. phagocytophilum DC organisms per PSGL-1 CHO cell by approximately fourfold to nearly that of CHO (−) cells (FIG. 8C). Anti-OmpA reduced bacterial binding to PSGL-1 CHO cells by approximately two-fold. Anti-OmpA23-40, anti-OmpA41-58, and preimmune serum had no effect. These results indicate that the OmpA binding domain lies within amino acids 59 to 74 and this region is important for A. phagocytophilum recognition of sLe^(x)-capped PSGL-1.

Molecular Docking Models of A. phagocytophilum OmpA-sLe^(x) Interactions Suggest that Residues within OmpA59-74 Engage sLe^(x)

To complement the antibody blocking experiments, molecular modeling and docking was used to identify the OmpA amino acids that possibly contact sLe^(x). First, a three-dimensional model of the invasin was generated. A crystal structure for A. phagocytophilum OmpA has yet to be determined, but an abundance of crystal structures for similar bacterial proteins have been determined. The Phyre2 (Protein Homology/Analogy Recognition Server version 2.0) server, which predicts three dimensional structures for protein sequences and threads the predicted models on known crystal structures (Kelley et al., 2009), was used to generate a tertiary structure model for OmpA (FIG. 9A). The resulting homology model predicted that OmpA residues 59 to 74 form part of a surface-exposed alpha helix (FIG. 9A), which could interact with ligands. Surface electrostatic values calculated using the adaptive Poisson-Boltzmann solver (APBS) plugin for PyMOL indicated that OmpA amino acids 19 to 74 have an overall cationic surface charge. The rest of the modeled protein exhibits an overall anionic surface charge (FIGS. 9C and D). These findings are consistent with prior observations that bacterial and viral proteins that interact with sLe^(x) and/or sialic acid do so at cationic surface patches (Chung et al., 2007; Hermans et al., 2012; Dormitzer et al., 2002; Stein et al., 1994; Varghese et al., 1992).

For docking predictions, the sLe^(x) glycan (FIG. 9B) was extracted from the crystal structure of sLe^(x)-capped PSGL-1 (DOI:10.2210/pdb1g1s/PDB). Autodock Vina was used to predict how OmpA might interact with sLe^(x). The search grid encapsulated OmpA19-74 (FIG. 9A). The top two docking models, each with the same predicted affinity value of −4.2 kcal/mol, displayed similar interactions between sLe^(x) and the OmpA region encompassed by amino acids 59 to 74. In both models, K64 of OmpA was predicted to bind the α2,3-sialic acid residue of sLe^(x) (FIGS. 9E and F). G61 was also predicted to interact with sLe^(x) in both models, though it was predicted to bind α2,3-sialic acid in one model and α1,3-fucose in the other. Lastly, K60 was predicted to bind the B1,3-galactose residue of sLe^(x) in the docking model presented in FIG. 9F. Together, the in silico predictions and peptide antibody blocking results suggest that OmpA59-74 contains critical residues that interact with sLe^(x) to promote A. phagocytophilum infection of host cells.

OmpA is Conserved Among A. phagocytophilum Strains and K64 is Conserved Among Anaplasmataceae OmpA Proteins

Aligning the OmpA sequence from the A. phagocytophilum NCH-1 strain, which was originally isolated from a HGA patient in Nantucket, Mass. (Kolbert et al., 1997), with those encoded by geographically diverse A. phagocytophilum isolates that had been recovered from infected humans, animals, and ticks revealed that OmpA is highly conserved among these strains (FIGS. 10A and B). Eight of the nine sequences were identical. The OmpA of NorV2 Norwegian sheep isolate had only three amino acid differences, none of which were within the binding domain encompassed by residues 59 to 74. The high degree of OmpA sequence conservation further supports the invasin's importance to A. phagocytophilum pathobiology. Next NCH-1 OmpA residues 19 to 74 were aligned with corresponding regions of OmpA homologs from A. marginale and Ehrlichia spp., which are in the family Anaplasmataceae with A. phagocytophilum and infect bovine erythrocytes and human and animal monocytes, respectively (Carylon et al., 2012; Mansueto et al., 2012; Suarez et al., 2011). A. phagocytophilum OmpA K64 that was predicted to potentially interact with sLe^(x) (FIG. 9, E and F), was the only binding domain residue that was conserved among all Anaplasmataceae OmpA regions examined (FIG. 10B). Additional residues within the A. phagocytophilum OmpA binding domain, including the other two predicted to interact with sLe^(x), K60 and G61 (FIG. 9, E and F), were conserved among Anaplasma spp. but not Ehrlichia spp. OmpA proteins (FIG. 10B).

G61 and K64 are Essential for Recombinant OmpA to Optimally Bind to Mammalian Host Cells and Competitively Inhibit A. phagocytophilum Infection

Because A. phagocytophilum is an obligate intracellular bacterium, developing a knock out-complementation system for this organism has proved challenging and has not been described. Therefore, a series of alternative approaches was utilized to further functionally evaluate OmpA. Recombinant OmpA can be used as a competitive agonist to block A. phagocytophilum access to its receptor and thereby inhibit infection. This phenomenon was exploited to further define the OmpA amino acids that are critical for receptor recognition and bacterial uptake by assessing the competitive agonist abilities of OmpA proteins having site-directed amino acid changes. This approach was built on the rationale that OmpA proteins in which the binding domain was disrupted would be unable to inhibit infection. First, OmpA proteins N-terminally fused to glutathione-S-transferase (GST) were generated, each of which had an insertion of the peptide CLNHL at one of six different sites within residues 19 to 78. This approach has been used in previous studies to disrupt proteins' binding domains without perturbing overall protein structure, and the insertion sequence that was devised for this purpose was a consensus of the insertion peptides used in those studies (Anton et al., 2004; Grande et al., 2007; Okoye et al., 2006). Incubating HL-60 cells with the positive control, GST-OmpA, prior to the addition of DC bacteria resulted in a significant reduction in the percentage of infected cells relative to GST alone (FIG. 11A). GST-OmpA proteins carrying insertions between residues 67 and 68 and between 62 and 63 were completely and partially abrogated, respectively, in their abilities to inhibit A. phagocytophilum infection. GST-OmpA proteins bearing insertions at other sites were unaffected in their ability to inhibit infection.

Next the specific amino acids of GST-OmpA that were critical for it to inhibit A. phagocytophilum infection were identified. The competitive agonist assay was repeated using GST-OmpA proteins in which select amino acids had been mutated to alanine (FIG. 10 and FIG. 11, B and C). Many of the targeted residues were within OmpA amino acids 59 to 74. R32 and D53 were selected because they lie outside of residues 59 to 74, and, accordingly, it was anticipated that substituting them would not alter OmpA function. GST-OmpAK64A was considerably reduced in its ability to inhibit A. phagocytophilum infection (FIG. 11, B and C), thereby indicating that this highly conserved residue was critical for GST-OmpA to serve as a competitive agonist. K65, however, was dispensable for this function, as the blocking ability of GST-OmpAK65A was uncompromised and the blocking ability of GST-OmpAKK6465AA was no greater than that of GST-OmpAK64. GST-OmpAG61A displayed a modest but significant decline in its competitive agonist ability. Replacement of both G61 and K64 with alanines yielded an additive effect that was greater than substituting either residue alone, as GST OmpAGK6164AA was unable to inhibit infection. GST-OmpA proteins in which R32, D53, K60, E69 and E72 had been mutated to alanine were each unaffected in the ability to hinder infection.

Given that K64 and G61 are vital and contributory, respectively, to the ability of recombinant OmpA to competitively inhibit A. phagocytophilum infection, it was evaluated if these residues mediate binding to mammalian host cell surfaces. RF/6A and HL-60 cells were incubated with His-tagged OmpA proteins. After unbound proteins were washed away, bound proteins were detected by flow cytometry using a His-tag antibody. His-tagged OmpA and OmpAD53A bound equally well to RF/6A cells (FIG. 11D). His-OmpAG61A bound poorly, His-OmpAK64A even more so, and His-OmpAGK6164AA could not bind to host cells. Collectively, these data are consistent with the invasin-receptor contacts predicted by the OmpA-sLe^(x) docking models and underscore the importance of OmpA K64 and G61 to OmpA-receptor interactions.

OmpA Interacts with α1,3-Fucose on Mammalian Host Cell Surfaces

α1,3-fucose is critical for A. phagocytophilum to bind PSGL-1-modeled glycopeptides, to bind and invade human and murine myeloid cells, and to establish infection in laboratory mice. Consistent with these observations, PSGL-1 CHO cells that had been pretreated with α1,3/4-fucosidase were approximately three-fold less permissive for A. phagocytophilum binding (FIG. 12A). Multiple lines of evidence led to the hypothesis that OmpA binds α1,3-fucose. First, OmpA binds α2,3-sialic acid, which is in close proximity to α1,3-fucose on sLe^(x). Second, the docking model in FIG. 9E predicted that OmpA residues within the binding domain contact both α2,3-sialic acid and α1,3-fucose of sLe^(x). Third, OmpA is important for A. phagocytophilum infection of not only myeloid, but also endothelial cells. Fourth, fucose residues are critical for the pathogen to invade RF/6A endothelial cells, as pretreatment of the host cells with α1,3/4-fucosidase made them significantly less permissive to A. phagocytophilum binding (FIG. 12B) and infection (FIG. 12C).

To determine if OmpA recognizes fucose, His-tagged OmpA was incubated with RF/6A cells that had been treated with α1,3/4-fucosidase and binding was assessed by immunofluorescence microscopy and flow cytometry. α2,3/6-sialidase-treated RF/6A cells were included as a positive control for a treatment that would make the host cells less permissive to recombinant OmpA binding. To verify the efficacy and specificity of both glycosidases, treated and untreated host cells were screened with AAL (Aleuria aurantia lectin) and MAL II (Maackia amurensis lectin II). AAL recognizes fucose residues that are in α1,3- and α1,6-linkages with N-acetylglucosamine (Yamashita et al., 1985; Chandrasekaran et al., 2003). MAL II detects sialic acids that are in α2,3-linkages with galactose. Fucosidase treatment abolished AAL but not MAL II binding, while sialidase treatment eliminated MAL II but not AAL binding (FIG. 13, A to D). Thus, the glycosidases were effective and specific. His-OmpA binding to both sialidase- and fucosidase-treated RF/6A cells was comparablyreduced relative to vehicle control treated cells, while binding of His-Asp14 was unaffected. Incubating the host cells with MAL II or AAL prior to the addition of His-OmpA competitively reduced the efficiency of His-OmpA binding by similar degrees as sialidase or fucosidase, respectively (FIG. 13E). Overall, these observations demonstrate that optimal adhesion of OmpA to host cells involves both α2,3-sialic acid and α1,3-fucose and that Asp14 utilizes neither sialic acid nor fucose to bind to host cells.

OmpA Interacts with 6-Sulfo sLe^(x) on RF/6A Endothelial Cell Surfaces

Because His-OmpA binding to RF/6A cells involved recognition of α2,3-sialic acid and α1,3-fucose (FIG. 13), it was hypothesized that OmpA interacts with sLe^(x) or a sLe^(x)-like receptor on these host cells. sLe^(x) and the sLe^(x)-like molecule, 6-sulfo sLe^(x) (Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)][HSO3(3-6)]GlcNAc1) (FIG. 14A) have both been detected on the surfaces of high endothelial venal, vascular, cancerous, and/or inflamed endothelial cells. To assess if either glycan is present on RF/6A cells, they were screened with sLe^(x) antibodies, CSLEX1 and KM93 and the 6-sulfo-sLe^(x) antibody, G72. Robust G72 signal but little to no CSLEX1 or KM93 signal was detected on RF/6A cells (FIG. 14, B and C). Binding of His-OmpA to RF/6A cells that had been pretreated with G72 was pronouncedly reduced relative to cells that had been incubated with CSLEX1, KM93, or isotype control antibody (FIG. 14D). Thus, A. phagocytophilum OmpA recognizes 6-sulfo-sLe^(x) on RF/6A endothelial cells.

OmpA-Coated Beads Bind to and are Internalized by Non-Phagocytic Endothelial Cells

The ability of recombinant OmpA to bind to non-phagocytic RF/6A endothelial cells (FIGS. 12-14), suggests that, in addition to functioning as an invasin, it may also exhibit adhesin activity. Furthermore, while OmpA on the A. phagocytophilum surface acts cooperatively with Asp14 and AipA to mediate bacterial binding to and invasion of mammalian host cells, its ability to mediate these processes by itself is unknown. Therefore, the ability of recombinant OmpA to confer adhesiveness and invasiveness to inert particles was assessed. His-OmpA was coupled to fluorescent microspheres that were 1.0 μm in diameter, a size similar to that of the diameter of an A. phagocytophilum DC organism (0.8±0.2 μm). Successful conjugation of His-OmpA to the beads was confirmed by immunofluorescence using OmpA antiserum (FIG. 15A). RF/6A cells were incubated with recombinant OmpA-coated or non-coated control beads and screened with OmpA antibody to determine the numbers of beads bound per cell. To assess bead uptake, the cells were incubated for an additional 1 to 8 h and trypsin was used to remove non-internalized beads prior to screening. Immunofluorescence microscopy revealed that significantly more OmpA coated beads bound to and were internalized by RF/6A cells versus control beads (FIG. 15, B and D). Scanning electron microscopy corroborated these results, as OmpA coated bead were observed bound to and inducing the formation of filopodia-like structures on the surfaces of RF/6A cells or covered by plasma membrane (FIG. 15C). Thus, OmpA alone was sufficient to mediate bead binding to and uptake by non-phagocytic RF/6A endothelial cells.

Binding and Uptake of OmpA-Coated Beads by Myeloid Cells is Dependent on sLe^(x)

Next the ability of His-OmpA coated beads to bind and enter HL-60 cells was assessed and, if so, whether these processes involve the OmpA myeloid cell receptor, sLe^(x). Scanning electron microscopy revealed that OmpA beads bound to and induced their own uptake into HL-60 cells (FIG. 16A). Relative to the results obtained using RF/6A cells (FIG. 15D), OmpA coated bead binding to HL-60 cells was reduced (FIG. 16B). However, of the OmpA beads that did bind, approximately half of them were internalized (FIG. 16C). Approximately three-fold fewer control beads than OmpA coated beads bound to and were taken in (FIG. 16, B and C). OmpA bead uptake, but not adherence was pronouncedly inhibited when the assay was performed at 4° C. versus 37° C. Beads coated with OmpAG61A, OmpAK64A, OmpAGK6164AA, and OmpAKK6465AA were significantly compromised in their abilities to bind to and be internalized by HL-60 cells (FIG. 16, B and C). OmpA bead cellular adherence and entry were significantly inhibited and neutralized, respectively, for host cells that had been pretreated with α2,3/6-sialidase or α1,3/4-fucosidase (FIG. 16, D and E). Moreover, the sLe^(x)-specific antibody, CSLEX1 significantly reduced binding and blocked internalization of OmpA beads into HL-60 cells (FIG. 16, F and G). KPL-1, an antibody that is specific for and blocks A. phagocytophilum binding to the PSGL-1 N-terminus, did not affect OmpA bead adherence or uptake (FIG. 16, H and I). These data indicate that OmpA coated beads bind and enter myeloid cells in a sLe^(x)-dependent manner and require OmpA residues G61 and K64 to optimally do so.

Delineation of the Asp14 Binding Domain

Of the three invasins that cooperatively function to facilitate A. phagocytophilum infection of mammalian host cells, only the binding domain of Asp14 had yet to be defined. Asp14 is a 124-amino acid (13.8 kDa) protein, and its binding domain lies within residues 101 to 124. To further narrow down this region, antisera were raised against residues 98 to 112 and 113 to 124. Both antisera recognized GST-Asp14, but not GST-Asp141-88 or GST alone. Also, antiserum targeting Asp1498-112 but not Asp14113-124 detected GST-Asp141-112 and each antiserum was specific for the peptide against which it had been raised. Next, the abilities of anti-Asp1498-112 and anti-Asp14113-124 to inhibit A. phagocytophilum infection of HL-60 cells were assessed. Incubating DC bacteria with Asp14113-124 antibody reduced the percentages of infected cells in a dose dependent manner, whereas Asp1498-112 antibody had no effect (FIG. 17A). When used together, antisera against Asp14113-124 and OmpA59-74 reduced A. phagocytophilum by approximately four-fold (FIG. 17B). The observed blocking effect was significantly greater than that achieved with either antiserum alone or when either was paired with antisera that targeted irrelevant regions of OmpA or Asp14. To ensure that the blocking effects achieved by the OmpA59-74 and Asp14113-124 antisera were specific, fragment antigen binding (Fab fragment) portions of OmpA23-40, OmpA41-48, OmpA59-74, Asp1498-112, Asp14113-124, or OmpA59-74 and Asp14113-124 antibodies were prepared and assessed for the ability to inhibit A. phagocytophilum infection of HL-60 cells. Consistent with results obtained using intact antibodies, OmpA59-74 Fab, Asp14113-124 Fab, and the combination thereof achieved the greatest reductions in the percentage of infected cells and morulae per cell (FIG. 17, C and D).

An Antisera Combination Targeting the OmpA, Asp14, and AipA Binding Domains Pronouncedly Inhibits A. phagocytophilum Infection of Host Cells

It was shown in Example 1 that a combination of antisera that had been raised against the entireties of OmpA, AipA, and Asp14 strongly inhibited A. phagocytophilum infection of mammalian host cells. To refine this blocking approach, DC organisms were treated with a cocktail of antibodies specific for OmpA59-74, Asp14113-124, and AipA9-21 prior to incubating the bacteria with HL-60 cells. This antibody combination significantly attenuated infection, reducing the percentage of infected cells and number of morulae per cell by approximately five-fold (FIG. 18, A and B). The reduction in infection achieved using the combination antisera was due to effective blocking of bacterial adhesion to HL-60 cell surfaces, as combination antisera specific for OmpA59-74, Asp14113-124, and AipA9-21 reduced the numbers of bound A. phagocytophilum organisms per cell by more than four-fold relative to the same amount of preimmune serum (FIG. 18C). The observed reductions in bacterial adhesion and infection achieved by targeting all three binding domains were greater than those achieved using (1) antibodies that targeted only one or two of the binding domains and (2) combinations of antibodies against one or two of the binding domains together with antibodies against irrelevant portions of OmpA, Asp14, or AipA. Thus, targeting the OmpA, Asp14, and AipA binding domains together produced a synergistic blocking effect that protects host cells from A. phagocytophilum infection.

Discussion

This study identified the OmpA and Asp14 binding domains and defined the OmpA residues that are critical for adhesion and invasion. The OmpA binding domain lies within amino acids 59 to 74 and it, like the rest of the protein, is highly conserved among A. phagocytophilum strains known to cause disease in humans and animals. Antibody against OmpA59-74 inhibited bacterial binding to PSGL-1 CHO cells and infection of HL-60 cells. OmpA59-74 is predicted to be a solvent exposed alpha helix and part of a cationic surface patch that binds sLe^(x), an interaction that is similar to those between staphylococcal superantigen-like (SSL) protein family members and sLe^(x). SSL4, SSL5, and SSL11 each use basic residues within cationic surface pockets to interact with α2,3-sialic acid of sLe^(x) (Chung et al., 2007; Hermans et al., 2012; Baker et al., 2007). Likewise, other pathogens' sialic acid binding proteins, including uropathogenic Escherichia coli sialic acid-specific S fimbrial adhesin (Morschhauser et al., 1990), pertussis toxin of Bordetella pertussis (Stein et al., 1994), influenza viral neuraminidase (Varghese et al., 1992), canine adenovirus 2 capsid protein (Rademacher et al., 2012), and rhesus rotovirus VP4 (Dormitzer et al., 2002) all use basic residues localized within cationic surface pockets to target sialic acid. The Asp14 binding domain is within amino acids 113 to 124. Antibody specific for Asp14113-124 abrogated bacterial binding and infection of host cells. As Asp14 bears no semblance to any known crystal structure, it could not be modeled. However, from the data presented herein it can be inferred that Asp14 amino acids 113 to 124 are exposed on the surfaces of A. phagocytophilum and the invasin itself.

OmpA K64 is essential for and G61 contributes to the ability of OmpA to bind to mammalian host cells. These experimental findings support the top two OmpA-sLe^(x) docking models, both of which predicted the involvement of K64 and G61 in interacting with α2,3-sialic acid and α1,3-fucose of sLe^(x). The actual interactions between OmpA and sLe^(x) are likely a hybrid of those predicted by the two docking models because, while both predicted the involvement of K64 and G61, one also predicted the involvement of K60, which was found to be negligible for OmpA to act as a competitive agonist. OmpA K64 and G61 may play functionally conserved roles among members of the family Anaplasmataceae and the genus Anaplasma. K64 is present in all Anaplasma and Ehrlichia spp. OmpA proteins, while G61 is conserved among Anaplasma but not Ehrlichia spp. OmpA proteins. A. marginale agglutinates bovine red blood cells in a sialidase-sensitive manner, indicating that it interacts with sialylated glycans on erythrocyte surfaces. E. chaffeensis OmpA contributes to infection of monocytic cells (Cheng et al., 2011). Compared to the conservation exhibited among Anaplasma spp. OmpA proteins, A. phagocytophilum and E. chaffeensis OmpA proteins are more divergent in sequence, especially in the binding domain, which may contribute to these pathogens' tropisms for different leukocytes. Still, because of its conservation, the E. chaffeensis OmpA residue that corresponds to A. phagocytophilum OmpA K64 may be involved in binding to a sLe^(x)-related glycan on monocytic cells.

Together with α2,3-sialic acid, α1,3-fucose is critical for A. phagocytophilum binding and infection. OmpA binds α1,3-fucose, as can be inferred from the observations that recombinant OmpA bound poorly to RF/6A endothelial cells from which α1,3/4-fucose residues had been removed or that had been incubated with the α1,3/6-fucose-specific lectin, AAL. The ability of OmpA to bind α2,3-sialic acid and α1,3-fucose is consistent with the close proximity of the two sugar residues to each other in sLe^(x) and related glycans and also with OmpA-sLe^(x) molecular docking predictions. Yet, RF/6A cells, which support A. phagocytophilum binding and infection, express very little to no sLe^(x). Rather, they express 6-sulfo-sLe^(x), which presents α2,3-sialic acid and α1,3-fucose in the same orientation and proximity to each other as sLe^(x). Recombinant OmpA binding to RF/6A cells was significantly reduced in the presence of 6-sulfo-sLe^(x) antibody, but not sLe^(x) antibodies, thereby supporting that 6-sulfo-sLe^(x) is an A. phagocytophilum receptor on these cells. Thus, A. phagocytophilum OmpA interacts with glycans that present α2,3-sialic acid and α1,3-fucose in a similar manner as sLe^(x).

OmpA by itself functions as both an adhesin and an invasin, as demonstrated by the ability of His-OmpA to confer adhesive and internalization capabilities to inert beads. Approximately half of the His-OmpA beads that bound to host cells were internalized, a degree of uptake that was similar to that reported for C. burnetii OmpA coated beads (Martinez et al., 2014). Twenty-fold more OmpA coated beads bound to RF/6A cells than to HL-60 cells. Similarly, recombinant OmpA binding to RF/6A cells but not to HL-60 cells could be detected by immunofluorescence microscopy and flow cytometry. Nonetheless, the ability of recombinant OmpA to competitively antagonize A. phagocytophilum binding and infection of HL-60 cells demonstrates its ability to bind to the host cells, but it apparently does so at too low an avidity to remain bound during the wash steps associated with sample preparation for the detection methods used. The observed differences in OmpA binding to HL-60 versus RF/6A cells could be due to differences in the levels of sLe^(x) and 6-sulfo-sLe^(x) on HL-60 and RF/6A cell surfaces or perhaps due to the presence of an additional, undefined OmpA receptor on RF/6A cells. Yet another possibility is that the bacterium binds with a greater avidity to 6-sulfo-sLe^(x) than to sLe^(x).

Because of the essential and cooperative roles that OmpA, Asp14, and AipA play in the A. phagocytophilum lifecycle, blocking their ability to function prevents both infection and bacterial survival. Moreover, directing the immune response to their binding domains enhances protective efficacy. In this study, an antibody cocktail specific for the OmpA, Asp14, and AipA binding domains blocked A. phagocytophilum infection of host cells. The relevance of this work extends to other obligate intracellular pathogens that use multiple invasins, including A. marginale, E. chaffeensis, spotted fever rickettsiae, Chlamydia spp., Mycobacterium spp., and Orientia tsutsugamushi, as their survival hinges on their abilities to enter host cells.

REFERENCES

-   Aguero-Rosenfeld M E, Donnarumma L, Zentmaier L, Jacob J, Frey M, et     al. (2002) Seroprevalence of antibodies that react with Anaplasma     phagocytophila, the agent of human granulocytic ehrlichiosis, in     different populations in Westchester County, N.Y. J Clin Microbiol     40: 2612-2615. -   Alhumaidan, H., Westley, B., Esteva, C., Berardi, V., Young, C. and     Sweeney, J. (2013). Transfusion-transmitted anaplasmosis from     leukoreduced red blood cells. Transfusion 53, 181-186. -   Al-Khedery B, Lundgren A M, Stuen S, Granquist E G, Munderloh U G,     et al. (2012) Structure of the type IV secretion system in different     strains of Anaplasma phagocytophilum. BMC Genomics 13: 678. -   Annen, K., Friedman, K., Eshoa, C., Horowitz, M., Gottschall, J. and     Straus, T. (2012). Two cases of transfusion-transmitted Anaplasma     phagocytophilum. American journal of clinical pathology 137,     562-565. -   Anton B P, Raleigh 858 EA (2004) Transposon-mediated linker     insertion scanning mutagenesis of the Escherichia coli McrA     endonuclease. J Bacteriol 186: 5699-5707. -   Baker H M, Basu I, Chung M C, Caradoc-Davies T, Fraser J D, et     al. (2007) Crystal structures of the staphylococcal toxin SSL5 in     complex with sialyl Lewis X reveal a conserved binding site that     shares common features with viral and bacterial sialic acid binding     proteins. J Mol Biol 374: 1298-1308. -   Bakken J S, Goellner P, Van Etten M, Boyle D Z, Swonger O L, et     al. (1998) Seroprevalence of human granulocytic ehrlichiosis among     permanent residents of northwestern Wisconsin. Clin Infect Dis 27:     1491-1496. -   Barbosa, A. S., Abreu, P. A., Neves, F. O., Atzingen, M. V.,     Watanabe, M. M., Vieira, M. L., et al. (2006). A newly identified     leptospiral adhesin mediates attachment to laminin. Infection and     immunity 74, 6356-6364. -   Bastidas R J, Elwell C A, Engel J N, Valdivia R H (2013) Chlamydial     intracellular survival strategies. Cold Spring Harb Perspect Med 3:     α010256. -   Brennan, M. J. and Shahin, R. D. (1996). Pertussis antigens that     abrogate bacterial adherence and elicit immunity. American journal     of respiratory and critical care medicine 154, S145-149. -   Cardwell, M. M. and Martinez, J. J. (2009). The Sca2 autotransporter     protein from Rickettsia conorii is sufficient to mediate adherence     to and invasion of cultured mammalian cells. Infection and immunity     77, 5272-5280. -   Carlyon, J. A. (2012) Establishing intracellular infection:     modulation of host cell functions (Anaplasmataceae). In     Intracellular Pathogens II: Rickettsiales, G. H. Palmer, A. Azad     (eds.). Washington, D.C., ASM Press. -   Carlyon, J. A., Akkoyunlu, M., Xia, L., Yago, T., Wang, T.,     Cummings, R. D., et al. (2003). Murine neutrophils require     alpha1,3-fucosylation but not PSGL-1 for productive infection with     Anaplasma phagocytophilum. Blood 102, 3387-3395. -   Carlyon J A, Chan W T, Galan J, Roos D, Fikrig E (2002) Repression     of rac2 mRNA expression by Anaplasma phagocytophila is essential to     the inhibition of superoxide production and bacterial proliferation.     J Immunol 169: 7009-7018. -   Centers for Disease Control and Prevention (2013). Notice to     readers: final 2012 reports of nationally notifiable infectious     diseases. MMWR. Morbidity and mortality weekly report 62, 669-682. -   Chan, Y. G., Cardwell, M. M., Hermanas, T. M., Uchiyama, T. and     Martinez, J. J. (2009). Rickettsial outer-membrane protein B (rOmpB)     mediates bacterial invasion through Ku70 in an actin, c-Cbl,     clathrin and caveolin 2-dependent manner. Cellular microbiology 11,     629-644. -   Chan, Y. G., Riley, S. P. and Martinez, J. J. (2010). Adherence to     and invasion of host cells by spotted Fever group rickettsia     species. Frontiers in microbiology 1, 139. -   Chandrasekaran E V, Chawda R, Rhodes J M, Locke R D, Piskorz C F, et     al. (2003) The binding characteristics and utilization of Aleuria     aurantia, Lens culinaris and few other lectins in the elucidation of     fucosyltransferase activities resembling cloned FT VI and apparently     unique to colon cancer cells. Carbohydr Res 338: 887-901. -   Chang, A., Kaur, R., Michel, L. V., Casey, J. R. and Pichichero, M.     (2011). Haemophilus influenzae vaccine candidate outer membrane     protein P6 is not conserved in all strains. Human vaccines 7,     102-105. -   Chang, B., Kura, F., Amemura-Maekawa, J., Koizumi, N. and     Watanabe, H. (2005). Identification of a novel adhesion molecule     involved in the virulence of Legionella pneumophila. Infection and     immunity 73, 4272-4280. -   Cheng Z, Miura K, Popov V L, Kumagai Y, Rikihisa Y (2011) Insights     into the CtrA regulon in development of stress resistance in     obligatory intracellular pathogen Ehrlichia chaffeensis. Mol     Microbiol 82: 1217-1234 -   Chung M C, Wines B D, Baker H, Langley R J, Baker E N, et al. (2007)     The crystal structure of staphylococcal superantigen-like protein 11     in complex with sialyl Lewis X reveals the mechanism for cell     binding and immune inhibition. Mol Microbiol 66: 1342-1355. -   Cirillo, S. L., Bermudez, L. E., El-Etr, S. H., Duhamel, G. E. and     Cirillo, J. D. (2001). Legionella pneumophila entry gene rtxA is     involved in virulence. Infection and immunity 69, 508-517. -   Dicko, A., Odusanya, O. O., Diallo, A. I., Santara, G., Barry, A.,     Dolo, A., et al. (2011). Primary vaccination with the 10-valent     pneumococcal non-typeable Haemophilus influenzae protein D conjugate     vaccine (PHiD-CV) in infants in Mali and Nigeria: a randomized     controlled trial. BMC public health 11, 882. -   Dormitzer P R, Sun Z Y, Wagner 814 G, Harrison S C (2002) The rhesus     rotavirus VP4 sialic acid binding domain has a galectin fold with a     novel carbohydrate binding site. EMBO J 21: 885-897. -   Duncan, C., Prashar, A., So, J., Tang, P., Low, D. E.,     Terebiznik, M. and Guyard, C. (2011). Lcl of Legionella pneumophila     is an immunogenic GAG binding adhesin that promotes interactions     with lung epithelial cells and plays a crucial role in biofilm     formation. Infection and immunity 79, 2168-2181. -   Emini, E. A., Hughes, J. V., Perlow, D. S. and Boger, J. (1985).     Induction of hepatitis A virus-neutralizing antibody by a     virus-specific synthetic peptide. J Virol 55, 836-839. -   Fukuda M, Spooncer E, Oates J E, Dell A, Klock J C (1984) Structure     of sialylated fucosyl lactosaminoglycan isolated from human     granulocytes. J Biol Chem 259: 10925-10935. -   Garduno, R. A., Garduno, E. and Hoffman, P. S. (1998).     Surface-associated hsp60 chaperonin of Legionella pneumophila     mediates invasion in a HeLa cell model. Infection and immunity 66,     4602-4610. -   Ge, Y. and Rikihisa, Y. (2007). Identification of novel surface     proteins of Anaplasma phagocytophilum by affinity purification and     proteomics. Journal of bacteriology 189, 7819-7828. -   Giufre, M., Carattoli, A., Cardines, R., Mastrantonio, P. and     Cerquetti, M. (2008). Variation in expression of HMW1 and HMW2     adhesins in invasive nontypeable Haemophilus influenzae isolates.     BMC microbiology 8, 83. -   Goodman, J. L., Nelson, C. M., Klein, M. B., Hayes, S. F. and     Weston, B. W. (1999). Leukocyte infection by the granulocytic     ehrlichiosis agent is linked to expression of a selectin ligand. The     Journal of clinical investigation 103, 407-412. -   Goodman J L, Nelson C, Vitale B, Madigan J E, Dumler J S, et     al. (1996) Direct cultivation of the causative agent of human     granulocytic ehrlichiosis. N Engl J Med 334: 209-215. -   Grande K K, Gustin J K, Kessler E, Ohman D E (2007) Identification     of critical residues in the propeptide of LasA protease of     Pseudomonas aeruginosa involved in the formation of a stable mature     protease. J Bacteriol 189: 3960-3968. -   Gribble D H (1969) Equine ehrlichiosis. J Am Vet Med Assoc 155:     462-469. -   Grimm, D., Tilly, K., Byram, R., Stewart, P. E., Krum, J. G.,     Bueschel, D. M., et al. (2004). Outer-surface protein C of the Lyme     disease spirochete: a protein induced in ticks for infection of     mammals. Proceedings of the National Academy of Sciences of the     United States of America 101, 3142-3147. -   Hao Q, Geng Z, Hou X X, Tian Z, Yang X J, et al. (2013)     Seroepidemiological investigation of lyme disease and human     granulocytic anaplasmosis among people living in forest areas of     eight provinces in China. Biomed Environ Sci 26:185-189. -   Hermans S J, Baker H M, Sequeira R P, Langley R J, Baker E N, et     al. (2012) Structural and functional properties of staphylococcal     superantigen-like protein 4. Infect Immun 80: 4004-4013. -   Herron, M. J., Nelson, C. M., Larson, J., Snapp, K. R.,     Kansas, G. S. and Goodman, J. L. (2000). Intracellular parasitism by     the human granulocytic ehrlichiosis bacterium through the P-selectin     ligand, PSGL-1. Science 288, 1653-1656. -   Hinnebusch, B. J., Perry, R. D. and Schwan, T. G. (1996). Role of     the Yersinia pestis hemin storage (hms) locus in the transmission of     plague by fleas. Science 273, 367-370. -   Hofmann, K. and Stoffel, W. (1993). TMbase-A database of membrane     spanning proteins segments. Biol. Chem. Hoppe-Seyler 374, 166. -   Hopkins, R. S., Jajosky, R. A., Hall, P. A., Adams, D. A.,     Connor, F. J., Sharp, P., Anderson, W. J., Fagan, R. F., Aponte, J.     J., Nitschke, D. A., Worsham, C. A., Adekoya, N. and     Chang, M. H. (2005) Summary of notifiable diseases United States,     2003. MMWR Morbidity and mortality weekly report 52, 1-85. -   Huang, B., Hubber, A., McDonough, J. A., Roy, C. R., Scidmore, M. A.     and Carlyon, J. A. (2010a). The Anaplasma phagocytophilum-occupied     vacuole selectively recruits Rab-GTPases that are predominantly     associated with recycling endosomes. Cellular microbiology 12,     1292-1307. -   Huang, B., Troese, M. J., Howe, D., Ye, S., Sims, J. T., Heinzen, R.     A., et al. (2010b). Anaplasma phagocytophilum APH_0032 is expressed     late during infection and localizes to the pathogen-occupied     vacuolar membrane. Microbial pathogenesis 49, 273-284. -   Huang, B., Ojogun, N., Ragland, S. A. and Carlyon, J. A. (2012)     Monoubiquitinated proteins decorate the Anaplasma     phagocytophilum-occupied vacuolar membrane. FEMS Immunology and     Medical Microbiology 64, 32-41. -   Jalalvand, F., Su, Y. C., Morgelin, M., Brant, M., Hallgren, O.,     Westergren-Thorsson, G., et al. (2013). Haemophilus influenzae     protein F mediates binding to laminin and human pulmonary epithelial     cells. The Journal of infectious diseases 207, 803-813. -   Jereb, M., Pecaver, B., Tomazic, J., Muzlovic, I., Avsic-Zupanc, T.,     Premru-Srsen, T., et al. (2012). Severe human granulocytic     anaplasmosis transmitted by blood transfusion. Emerging infectious     diseases 18, 1354-1357. -   Johnson R C, Kodner C, Jarnefeld J, Eck D K, Xu Y (2011) Agents of     human anaplasmosis and Lyme disease at Camp Ripley, Minn. Vector     Borne Zoonotic Dis 11: 1529-1534. -   Jurcisek, J. A., Bookwalter, J. E., Baker, B. D., Fernandez, S.,     Novotny, L. A., Munson, R. S., Jr. and Bakaletz, L. O. (2007). The     PilA protein of non-typeable Haemophilus influenzae plays a role in     biofilm formation, adherence to epithelial cells and colonization of     the mammalian upper respiratory tract. Molecular microbiology 65,     1288-1299. -   Kahlon, A., Ojogun, N., Ragland, S. A., Seidman, D., Troese, M. J.,     Ottens, A. K., et al. (2013). Anaplasma phagocytophilum Asp14 is an     invasin that interacts with mammalian host cells via its C terminus     to facilitate infection. Infection and immunity 81, 65-79. -   Karakantza M, Gibson F M, Cavenagh J D, Ball S E, Gordon M Y, et     al. (1994) SLe(x) expression of normal CD34 positive bone marrow     haemopoietic progenitor cells. Br J Haematol 86: 883-886. -   Kelley L A, Sternberg M J (2009) Protein structure prediction on the     Web: a case study using the Phyre server. Nat Protoc 4: 363-371. -   Kolbert C P, Bruinsma E S, Abdulkarim A S, Hofmeister E K, Tompkins     R B, et al. (1997) Characterization of an immunoreactive protein     from the agent of human granulocytic ehrlichiosis. J Clin Microbiol     35: 1172-1178. -   Krogh, A., Larsson, B., von Heijne, G. and Sonnhammer, E. L. (2001).     Predicting transmembrane protein topology with a hidden Markov     model: application to complete genomes. Journal of molecular biology     305, 567-580. -   Kyte, J. and Doolittle, R. F. (1982). A simple method for displaying     the hydropathic character of a protein. Journal of molecular biology     157, 105-132. -   Li F, Wilkins P P, Crawley S, Weinstein J, Cummings R D, et     al. (1996) Post translational modifications of recombinant     P-selectin glycoprotein ligand-1 required for binding to P- and     E-selectin. The Journal of biological chemistry 271: 3255-3264. -   Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene     expression data using real-time quantitative PCR and the 2(-Delta     Delta C(T)) Method. Methods 25, 402-408. -   Madigan J E, Gribble D (1987) Equine ehrlichiosis in northern     California: 49 cases (1968-1981). J Am Vet Med Assoc 190: 445-448. -   Mansueto P, Vitale G, Cascio A, Seidita A, Pepe I, et al. (2012) New     insight into immunity and immunopathology of Rickettsial diseases.     Clin Dev Immunol 2012: 967852. -   Martinez E, Cantet F, Fava L, Norville I, Bonazzi M (2014)     Identification of OmpA, a Coxiella burnetii Protein Involved in Host     Cell Invasion, by Multi-Phenotypic High-Content Screening. PLoS     Pathog 10: e1004013 -   Martinez, J. J. and Cossart, P. (2004). Early signaling events     involved in the entry of Rickettsia conorii into mammalian cells.     Journal of cell science 117, 5097-5106. -   Massung R F, Levin M L, Munderloh U G, Silverman D J, Lynch M J, et     al. (2007) Isolation and propagation of the Ap-Variant 1 strain of     Anaplasma phagocytophilum in a tick cell line. J Clin Microbiol 45:     2138-2143. -   Mastronunzio, J. E., Kurscheid, S. and Fikrig, E. (2012).     Postgenomic analyses reveal development of infectious Anaplasma     phagocytophilum during transmission from ticks to mice. Journal of     bacteriology 194, 2238-2247. -   Miller D P, McDowell J V, Bell J K, Marconi R T (2011)     Crystallization of the factor H-binding protein, FhbB, from the     periopathogen Treponema denticola. Acta Crystallogr Sect F Struct     Biol Cryst Commun 67: 678-681. -   Minnick M F, Raghavan R (2012) Developmental biology of Coxiella     burnetii. Adv Exp Med Biol 984: 231-248. -   Molleken, K., Becker, E. and Hegemann, J. H. (2013). The Chlamydia     pneumoniae invasin protein Pmp21 recruits the EGF receptor for host     cell entry. PLoS pathogens 9, e1003325. -   Molleken, K., Schmidt, E. and Hegemann, J. H. (2010). Members of the     Pmp protein family of Chlamydia pneumoniae mediate adhesion to human     cells via short repetitive peptide motifs. Molecular microbiology     78, 1004-1017. -   Morschhauser J, Hoschutzky H, Jann K, Hacker J (1990) Functional     analysis of the sialic acid-binding adhesin SfaS of pathogenic     Escherichia coli by site-specific mutagenesis. Infect Immun 58:     2133-2138. -   Nelson, C. M., Herron, M. J., Felsheim, R. F., Schloeder, B. R.,     Grindle, S. M., Chavez, A. O., et al. (2008). Whole genome     transcription profiling of Anaplasma phagocytophilum in human and     tick host cells by tiling array analysis. BMC Genomics 9, 364. -   Ojogun N, Barnstein B, Huang B, Oskeritzian C A, Homeister J W, et     al. (2011) Anaplasma phagocytophilum infects mast cells via     alpha1,3-fucosylated but not sialylated glycans and inhibits     IgE-mediated cytokine production and histamine release. Infect Immun     79: 2717-2726. -   Ojogun, N., Kahlon, A., Ragland, S. A., Troese, M. J.,     Mastronunzio, J. E., Walker, N. J., et al. (2012). Anaplasma     phagocytophilum outer membrane protein A interacts with sialylated     glycoproteins to promote infection of mammalian host cells.     Infection and immunity 80, 3748-3760. -   Okoye M E, Sexton G L, Huang E, McCaffery J M, Desai P (2006)     Functional analysis of the triplex proteins (VP19C and VP23) of     herpes simplex virus type 1. J Virol 80: 929-940. -   Park, J., Kim, K. J., Grab, D. J. and Dumler, J. S. (2003).     Anaplasma phagocytophilum major surface protein-2 (Msp2) forms     multimeric complexes in the bacterial membrane. FEMS microbiology     letters 227, 243-247. -   Perry, R. D. and Fetherston, J. D. (1997). Yersinia pestis—etiologic     agent of plague. Clin Microbiol Rev 10, 35-66. -   Pinne, M., Choy, H. A. and Haake, D. A. (2010). The OmpL37     surface-exposed protein is expressed by pathogenic Leptospira during     infection and binds skin and vascular elastin. PLoS neglected     tropical diseases 4, e815. -   Rademacher C, Bru T, McBride R, Robison E, Nycholat 946 C M, et     al. (2012) A Siglec like sialic-acid-binding motif revealed in an     adenovirus capsid protein. Glycobiology 22: 1086-1091. -   Reneer, D. V., Kearns, S. A., Yago, T., Sims, J., Cummings, R. D.,     McEver, R. P. and Carlyon, J. A. (2006). Characterization of a     sialic acid- and P-selectin glycoprotein ligand-1-independent     adhesin activity in the granulocytotropic bacterium Anaplasma     phagocytophilum. Cellular microbiology 8, 1972-1984. -   Reneer, D. V., Troese, M. J., Huang, B., Kearns, S. A. and     Carlyon, J. A. (2008). Anaplasma phagocytophilum PSGL-1-independent     infection does not require Syk and leads to less efficient AnkA     delivery. Cellular microbiology 10, 1827-1838. -   Rikihisa Y, Zhi N, Wormser G P, Wen B, Horowitz H W, et al. (1997)     Ultrastructural and antigenic characterization of a granulocytic     ehrlichiosis agent directly isolated and stably cultivated from a     patient in New York state. J Infect Dis 175: 210-213. -   Riley, S. P., Goh, K. C., Hermanas, T. M., Cardwell, M. M.,     Chan, Y. G. and Martinez, J. J. (2010). The Rickettsia conorii     autotransporter protein Sca1 promotes adherence to nonphagocytic     mammalian cells. Infection and immunity 78, 1895-1904. -   Sarkar, M., Reneer, D. V. and Carlyon, J. A. (2007). Sialyl-Lewis     x-independent infection of human myeloid cells by Anaplasma     phagocytophilum strains HZ and HGE1. Infection and immunity 75,     5720-5725. -   Seidman D, Ojogun N, Walker N J, Mastronunzio J, Kahlon A, et     al. (2014) Anaplasma phagocytophilum surface protein AipA mediates     invasion of mammalian host cells. Cell Microbiol 16: 1133-1145. -   Singh, B., Al-Jubair, T., Morgelin, M., Thunnissen, M. M. and     Riesbeck, K. (2013). The unique structure of Haemophilus influenzae     protein E reveals multiple binding sites for host factors. Infection     and immunity 81, 801-814. -   Sperandio M (2006) Selectins and glycosyltransferases 747 in     leukocyte rolling in vivo. FEBS J 273: 4377-4389. -   Stein P E, Boodhoo A, Armstrong G D, Heerze L D, Cockle S A, et     al. (1994) Structure of a pertussis toxin-sugar complex as a model     for receptor binding. Nat Struct Biol 1: 591-596. -   Stone, B. J. and Abu Kwaik, Y. (1998). Expression of multiple pili     by Legionella pneumophila: identification and characterization of a     type IV pilin gene and its role in adherence to mammalian and     protozoan cells. Infection and immunity 66, 1768-1775. -   Stuen S, Granquist E G, Silaghi C (2013) Anaplasma phagocytophilum—a     widespread multi-host pathogen with highly adaptive strategies.     Front Cell Infect Microbiol 3: 31. -   Suarez C E, Noh S (2011) Emerging perspectives in the research of     bovine babesiosis and anaplasmosis. Vet Parasitol 180: 109-125. -   Symington F W, Hedges D L, Hakomori S (1985) Glycolipid antigens of     human polymorphonuclear neutrophils and the inducible HL-60 myeloid     leukemia line. J Immunol 134: 2498-2506. -   Tilly, K., Krum, J. G., Bestor, A., Jewett, M. W., Grimm, D.,     Bueschel, D., et al. (2006). Borrelia burgdorferi OspC protein     required exclusively in a crucial early stage of mammalian     infection. Infection and immunity 74, 3554-3564. -   Troese, M. J. and Carlyon, J. A. (2009). Anaplasma phagocytophilum     dense-cored organisms mediate cellular adherence through recognition     of human P-selectin glycoprotein ligand 1. Infection and immunity     77, 4018-4027. -   Troese, M. J., Kahlon, A., Ragland, S. A., Ottens, A. K., Ojogun,     N., Nelson, K. T., et al. (2011). Proteomic analysis of Anaplasma     phagocytophilum during infection of human myeloid cells identifies a     protein that is pronouncedly upregulated on the infectious     dense-cored cell. Infection and immunity 79, 4696-4707. -   Truchan, H. K., Seidman, D. and Carlyon, J. A. (2013). Breaking in     and grabbing a meal: Anaplasma phagocytophilum cellular invasion,     nutrient acquisition, and promising tools for their study. Microbes     and infection/Institut Pasteur. -   Vandersmissen, L., De Buck, E., Saels, V., Coil, D. A. and Anne, J.     (2010). A Legionella pneumophila collagen-like protein encoded by a     gene with a variable number of tandem repeats is involved in the     adherence and invasion of host cells. FEMS Microbiol Lett 306,     168-176. -   Varghese J N, McKimm-Breschkin J L, Caldwell J B, Kortt A A, Colman     P M (1992) The structure of the complex between influenza virus     neuraminidase and sialic acid, the viral receptor. Proteins 14:     327-332. -   Verma, A., Brissette, C. A., Bowman, A. A., Shah, S. T.,     Zipfel, P. F. and Stevenson, B. (2010). Leptospiral endostatin-like     protein A is a bacterial cell surface receptor for human     plasminogen. Infection and immunity 78, 2053-2059. -   Wang, Y., Berg, E. A., Feng, X., Shen, L., Smith, T.,     Costello, C. E. and Zhang, Y. X. (2006). Identification of     surface-exposed components of MOMP of Chlamydia trachomatis     serovar F. Protein science: a publication of the Protein Society 15,     122-134. -   Xia, L., Ramachandran, V., McDaniel, J. M., Nguyen, K. N.,     Cummings, R. D. and McEver, R. P. (2003). N-terminal residues in     murine P-selectin glycoprotein ligand-1 required for binding to     murine P-selectin. Blood 101, 552-559. -   Yago, T., Leppanen, A., Carlyon, J. A., Akkoyunlu, M., Karmakar, S.,     Fikrig, E., et al. (2003). Structurally distinct requirements for     binding of P-selectin glycoprotein ligand-1 and sialyl Lewis x to     Anaplasma phagocytophilum and P-selectin. The Journal of biological     chemistry 278, 37987-37997. -   Yamashita K, Kochibe N, Ohkura T, Ueda I, Kobata A (1985)     Fractionation of L fucose-containing oligosaccharides on immobilized     Aleuria aurantia lectin. J Biol Chem 260: 4688-4693. -   Zhang J Z, Popov V L, Gao S, Walker D H, Yu X J (2007) The     developmental cycle of Ehrlichia chaffeensis in vertebrate cells.     Cell Microbiol 9: 610-618. -   Zhang, L., Zhang, C., Ojcius, D. M., Sun, D., Zhao, J., Lin, X., et     al. (2012). The mammalian cell entry (Mce) protein of pathogenic     Leptospira species is responsible for RGD motif-dependent infection     of cells and animals. Molecular microbiology 83, 1006-1023. -   Zhang S, Hai R, Li W, Li G, Lin G, et al. (2009) Seroprevalence of     human granulocytotropic anaplasmosis in central and southeastern     China. Am J Trop Med Hyg 81: 293-295. -   Zhang X C, Zhang L X, Li W H, Wang S W, Sun Y L, et al. (2012)     Ehrlichiosis and zoonotic anaplasmosis in suburban areas of Beijing,     China. Vector Borne Zoonotic Dis 12: 932-937.

While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein. 

I claim:
 1. An immunogenic composition, wherein said composition comprises a first polypeptide that is or includes one or more copies of SEQ ID NO:14, at least one second polypeptide that is or includes one or more copies of SEQ ID NO:03 and SEQ ID NO:06, and a vehicle or carrier suitable for administration to a subject, or wherein said composition comprises at least one third polypeptide that includes one or more copies of SEQ ID NO:14 and one or more copies of at least one of SEQ ID NO:03 and SEQ ID NO:06, and a vehicle or carrier suitable for administration to a subject, and wherein said first and third polypeptide does not include SEQ ID NO:
 13. 2. The immunogenic composition of claim 1, wherein at least one of said first, second, or third polypeptides is linked to one or more of: an amino acid spacer; an amino acid linker; a signal sequence; a stop transfer sequence; a transmembrane domain; a protein purification ligand; a heterologous protein; or one or more additional polypeptides comprising SEQ ID NO:01, 02, 03, 04, 05, 06, 07, 08, 09, 13, or
 15. 3. The immunogenic composition of claim 2, wherein at least one of said first, second, or third polypeptides is linked to said protein purification ligand and said protein purification ligand is selected from the group consisting of glutathione S-transferase (GST) and a histidine tag (His tag).
 4. A chimeric protein comprising a polypeptide that is or includes one or more copies of SEQ ID NO:14 and one or more copies of one or more polypeptide sequences, wherein said one or more polypeptide sequences is or includes at least one of SEQ ID NO:03 and SEQ ID NO:06 and wherein said polypeptide does not include SEQ ID NO:
 13. 